The Physical Environment of Chloroplasts as Related to their Morphology and Activity in Vitro

McClendon, John H.

doi:10.1104/pp.29.5.448

Cited by 38 publications

(4 citation statements)

References 29 publications

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“…in addition to the use of very low pressures, PEG 4000 was included in the isolation buffer. This polymer has been shown to stabilize chloroplasts of red and green algae during a purification procedure [ 37 ] or isolated thylakoid membranes during a temperature treatment [ 42 ]. Furthermore, PEG increases the viscosity of the isolation buffer, which likely reduces the shearing forces during the passage through the French Press.…”

Section: Resultsmentioning

confidence: 99%

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

et al. 2017

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BackgroundThe preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction.ResultsSpectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to diatoxanthin and diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants.ConclusionsThe present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1154-8) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

et al. 2017

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“…Creswell (7) Carbowax has the following known effects: A, it increases the osmotic pressure (15) ; B, it protects against tannins (6), and C, it precipitates proteins (20). For the previously reported osmotic pressure effects a high concentration of Carbowax was used.…”

Section: Methodsmentioning

confidence: 99%

Demonstration of Photophosphorylation by Maize Chloroplasts

Miflin

Hageman

1963

Plant Physiol.

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Photoplhosphorylation by isolate(i chlioroplasts has been reportedl and studied in several species (21), but no publislhed evidence of the reaction by maize chlioroplasts coul(l )e foundl. In a stu(ly of this reaction by maize chlioroplasts initial experiments using existing isolation techniques (2,12) (90 v). The homogenate was then filtered through eight layers -of cheesecloth and centrifuged at 2,000 X g for 7 minutes. The pellet was resuspended in 10 ml of grinding medium and recentrifuged at the same speed. The chloroplast pellet was finallv taken up in 1.5 ml of grinding medium. Although this procedure was satisfactory for spinach tisstue, mlaize chloroplasts isolated by the i(lentical

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“…Carbowax 4000 was first used by -McClendon (12) for isolating active chloroplasts from red algae and leaves of higher plants. He …”

Section: Methodsmentioning

confidence: 99%

Methods for the Extraction of Enzymes from Cereal Leaves with Especial Reference to the Triosephosphate Dehydrogenases.

Hageman¹,

Waygood²

1959

Plant Physiol.

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The Physical Environment of Chloroplasts as Related to their Morphology and Activity in Vitro

Cited by 38 publications

References 29 publications

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

Demonstration of Photophosphorylation by Maize Chloroplasts

Methods for the Extraction of Enzymes from Cereal Leaves with Especial Reference to the Triosephosphate Dehydrogenases.

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