Cold Storage of Isolated Class C Chloroplasts

Farkas, Daniel L.; Malkin, Shmuel

doi:10.1104/pp.64.6.942

Cited by 35 publications

(19 citation statements)

References 11 publications

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“…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.…”

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confidence: 88%

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Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

Eichacker¹,

Edhofer²,

Wanner³

1996

Plant Physiology

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Methods for the cryopreservation of protein import and integration in pea chloroplasts and of protein import or protein synthesis in tobacco mitochondria were modified to yield enzymatically active cryopreserved etioplasts from barley (Hordeum vurgare 1.). The cryoprotectants ethylene glycol and dimethy sulfoxide were about 64 and 77% effective, respectively, for the cryopreservation of etioplast intactness. Phototransformation of protochlorophyllide a, esterification of chlorophyllide a or zinc-pheophorbide a, and stabilization of the de novo synthesized plastid-encoded chlorophyllapoproteins P700, CP47, CP43, D2, and D1 were successfully preserved in liquid nitrogen. Cryopreservation of freshly prepared intact etioplasts completely retained enzymatic activities for accumulation of chlorophyll a or resulted in a slightly decreased yield of zinc-pheophytin a.Etioplasts isolated from 4-d-old dark-grown barley (Hordeum vulgare L.) seedlings are an ideal in vitro system to study the Chl-dependent accumulation of higher plant photosystems. In barley, etioplasts are formed in the absence of light from proplastids during the developmental phase of early primary leaf and plastid development (Robertson and Laetsch, 1974). Etioplasts accumulate PChlide, a Chl precursor, which is reduced to Chlide by PChlideoxidoreductase in an NADPH-dependent reaction in the light (Apel et al., 1980). Chlide is then esterified to GGPP by Chl-synthase in a light-independent step to yield Chl (Riidiger et al., 1980).In the absence of Chl, etioplasts accumulated neither Chl-Ps (Herrmann et al., 1985;Klein and Mullet, 1986) nor nuclear-encoded Chl alb-binding apoproteins (Apel, 1979;Bennett et al., 1984). The plastid-encoded Chl-Ps were shown to be translated and degraded at high rates . During de novo synthesis of Chl from its precursors, Chlide and GGPP, Chl-Ps were shown to be stabilized against proteolytic digestion (Eichacker et al., 1990;Kim et al., 1994).To further our understanding of the influence of Chl synthesis on the regulation of translation and Chl-P accumulation, it was necessary to prepare etioplasts by numerous time-consuming isolation steps. However, the highest activities of Chl-synthase and translation of Chl-P were obtained only when etioplasts were prepared immediately before use. These constraints limited the number of parallel experiments that could be performed in a series. We therefore had to develop a method for the rapid and reliable cryopreservation of barley etioplasts that retained the enzymatic activities required for the stabilization of the Chl-P.Severa1 compounds, such as DMSO, EG, and glycerol, have been reported to act as cryoprotective agents on plant tissues (Finkle et al., 1985;Chen and Li, 1989). 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). Her...

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mentioning

confidence: 88%

“…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).…”

Section: Cryopreservation Of Etioplast Lntactnessmentioning

confidence: 99%

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

Eichacker¹,

Edhofer²,

Wanner³

1996

Plant Physiology

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“…Among them, DMSO has long been known as one of the most effective cryoprotectants for both plant and animal tissues (13); ethylene glycol, glycerol, and glycine have also been documented as good cryoprotective agents in some cases (12,13). Here, these cryoprotectants at different concentrations were examined for their ability to preserve intact pea chloroplasts.…”

Section: Results and Discussion Preservation Of Isolated Intact Chlormentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Yuan¹,

Cline²,

Theg³

1991

Plant Physiol.

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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.

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Control of Photosynthetic Electron Transfer from the Reaction Center to Electron Carriers of Photosystem II Studied by Fluorescence Induction

Malkin

1981

Israel Journal of Chemistry

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Abstract. The relationships between the primary electron acceptor (0) and the secondary pool (AI) are examined from fluorescence induction curves, at various illumination times and relaxation dark times. The fast-phase in the fluorescence rise curve, obtained after insufficient relaxation time, indicates the occurrence of "unconnected" 0, i.e. the reaction between 0-and AI is blocked. Various treatments bring about similar disconnection: high pH, ethylene glycol, low temperature (all reversible), trypsin and reduction of A I by dithionite. All these treatments are similar to DCMU treatment. A scheme is suggested in which 0 is mostly unconnected in the dark and becomes immediately connected as it is reduced by light. The above treatments inhibit the reconnection reaction in the light. The thermodynamic equilibrium between the 0 populations that can or cannot be reconnected was analyzed from the fluorescence curves at various temperatures. The connection-disconnection switching reaction is probably controlled by conformational changes of an associated protein. This scheme explains the difference of apparent redox equilibria of 0 and A in the light or in the dark, as well as the lOP wave in the fluorescence induction of intact cells.

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Cold Storage of Isolated Class C Chloroplasts

Cited by 35 publications

References 11 publications

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

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

Control of Photosynthetic Electron Transfer from the Reaction Center to Electron Carriers of Photosystem II Studied by Fluorescence Induction

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