Chloroplast isolation procedure

The rate of photosynthesis was measured using a portable IRGA (ADC, England). Chloroplast isolation and measurements of photochemical activity which included PSII, PSI and whole chain electron transport activities, were carried out according to the procedure described by Sayeed and Mohanty (7). Estimation of chlorophyll was carried out following Arnon (8) and soluble leaf proteins were estimated following Lowry et al (9). 

Plant Material. Plants of Pisum sativum (L. cv. F tham First) and Phaseolus vulgaris (L. cv. Blue Lake) were grown and class D chloroplasts isolated according to procedures presented ewhere (5). 

Plant material (Spinach and Peperomia metallica) was obtained from laboratory cultures. Chloroplast isolation and preparation were done following procedures, described before (Schapendonk, 1980). Absorbance difference measurements were done with an instrumentation and computer-assisted processing as described elsewhere (van Kooten et al., 1983). Potential measurement in Peperomia chloroplasts by implanted micro-capillary electrodes, were also as described before (Schapendonk, 1980). 

Under the conditions where D. bardawil accumulates large amounts of 3-carotene, the carotene is concentrated within lipoidal globules located exclusively in the interthylakoid space within the algal chloroplast. A procedure was developed for the isolation and purification of these globules. They were found to contain over 60% 3-carotene with the rest being mostly neutral lipids (Fried et al, 1982). The 3-carotene was composed of approximately equal amounts of the all-trans and the 9-cis isomers. 

Isolation and comparisons of nucleic acids are being carried out on an ever increasing number of plant species. For many of these taxa, the procedures described previously for nuclear DNA,1>2 chloroplast DNA,3 and mitochondrial DNA4-6 have been invaluable. An additional, new source detailing many aspects of nucleic acid comparisons is highly recommended.7 This chapter describes protocols that we have found useful with plants containing many secondary metabolites. In addition, it summarizes procedures from other laboratories that may be helpful in future studies involving nucleic acid comparisons. 

Aminoacid-tRNA synthetases were isolated from spinach chloroplasts by methods developed at Jealott s Hill. Cytoplasmic aaTRS s from carrot were isolated by standard procedures. Compounds were assayed as inhibitors of the chloroplastic aaTRS s at a single rate, 10 pM. The reaction was followed by phosphate generation and the percentage inhibition determined using literature methods. The IC50 value for the spinach chloroplastic enzymes and inhibition data for carrot cytoplasmic aaTRS s were determined for compounds of interest. All compounds were tested for post-emergence herbicidal activity, at 0.125, or at 0.5, or at 2.0 k a in standard screens." ... 

Between photosystem II submembrane fractions which must be isolated and whole cells which must be cultivated, many immobilization works (28 on 45 references cited) employed chloroplasts and thylakoids intact chloroplasts, mixtures of chloroplasts and photosynthetic membranes, or specially, thylakoids alone. To obtain these photosynthetic membranes, the chloroplasts were subjected to osmotic shock by briefly placing them in a hypotonic medium. This procedure ruptures the chloroplast envelope and releases the stroma. The stripped chloroplasts were then returned to an isotonic medium. The interest of this photosynthetic material is justified by a direct contact between the reaction sites and the operation medium. The photosyntheric preparations were often obtained from spinach leaves. 

This procedure has been used with success on a wide variety of plant groups and even some animals. The method is used to isolate total genomic DNA (nuclear, chloroplast, and mitochondrial). It is a rapid, inexpensive method that is suitabie for use in conjunction with other protocois, such as isolation of DNA enriched for cpDNA. it is also easy to scale down for use in population sampling, using 0.01 g or less of fresh tissue. Other applications include isolation of DNA from herbarium specimens (Doyle Dickson, 1987. Taxon 36 715-722), and isolation of RNA. A brief word on the history of the protocol is in order. This procedure was modified by us (Doyle and Doyle, 1987. Phytochemical Bulletin 19 11-15) for use with fresh piant tissue from a method of Saghai-Maroof et al. (1984, PNAS USA 81 8014-8019) who used lyophilized tissue. They in turn had developed their procedure from earlier protocols. We were recently asked to publish a slightly modified version of our procedure (Doyle and Doyle, 1990 Focus 12 13-15). We recently learned from Brian Taylor (Texas A M University, USA) that he had published a virtually identical procedure for fresh tissue, also in Focus, in 1982 (Taylor Powell, Focus 4 4-6) of which we (and apparently the editors of Focus ) were entirely unaware. It is indeed a useful procedure, thus independently confirmed. 

Discard supernatant. Separately resuspend chloroplast and nuclear pellets In 5-7 ml 60°C 2x CTAB buffer and proceed as in Doyle Doyle (1987) CTAB procedure to isolate cp and nuclear DNAs. 

Native PS I particles (Chl/P700 ratio - 200) were isolated from spinach chloroplasts following the procedure of Mullet et. al. [5,6]. 

We have therefore applied the Li Cl extraction procedure on coupled spinach thylakoids, in an attempt to isolate CFi/3 by the method used for Rr/ and compare their activities. In the following experiments the composition and activities of the chloroplast LiCl extract and of a CFxi enriched fraction are described. 

The Fi portion of the ATP synthase has three copies of the P subunit which is thought to contain the nucleotide-binding/catalytic sites of the enzyme. We recently described a procedure for isolating this important subunit from spinach chloroplast coupling factor 1 (CFi) and showed that it was able to restore both ATP hydrolysis and a low rate of ATP synthesis to chromatophores of Rhodospirillum rubrum which lacked the native P subunit. ... 

Broken pea chloroplasts were isolated according to a modified procedure used for spinach chloroplasts [1]. Chloroplast were resuspended in 2 mM MgCl2, 0.5 mg/ml BSA and 5 mM HEPES/KOH pH 7.5 with either 200 mM Sorbitol (low... 

The procedure for the isolation of chloroplasts from cell wall mutants of Chlamydomonas yields relatively pure, active chloroplasts. These chloroplast were photosynthetically active (3) and synthesized proteins in organello (4). They also imported, processed and assembled in vitro synthesized precursors for the small subunit of ribulose bisphosphate carboxylase and for the light harvesting chlorophyll a/b proteins (5, 6, 7). 

Tomato (Lycopersicon esculenturn cv Platense) chloroplasts (9) and chromoplasts (3) were isolated by previously published procedures. Pt DNA and total RNA were prepared as described in Ref. 3 and 9, respectively. Conditions for agarose electrophoresis of DNA restriction fragments, electrophoresis of RNA in formaldehyde/formamide/agarose gels Southern and Northern hybridizations were esentially those of Ref. 10. 

Preparation of partially purified stromal ATI. Flats of plants were placed in darkness for 1-2 days prior to harvesting leaves in order to deplete starch grains and therefore maximize the yield of intact chloroplasts. Enzymes were purified from 0.4 - 2 kg of expanding leaves from young plants, essentially as described previously by Bertrams and Heinz (1981), except that chloroplasts were isolated from leaves using the procedure of Nakatani and Barber (1977). 

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