Chloroplast isolation

Ozone causes both quantitative and qualitative changes in carbon dioxide fixation patterns. Wilkinson and Bames, using carbon dioxide-found a reduction in radioactivity in soluble sugars and increases in free amino acids and sugar phosphates in white pine after a 10-min exposure to ozone at 0.10 ppm. Miller observed a decrease in carbon dioxide-fixation in ponderosa pines that correlated with loss of chlorophyll, after exposure to ozone at 0.30-0.35 ppm. The Hill reaction rates of chloroplasts isolated from healthy and ozone-injured ponderosa pine indicated that both light and dark reactions of the chloroplasts from ozone-injured plants were depressed. Barnes found depressed photosynthesis and stimulated respiration in seedlings of four pine species of the southeastern United States after exposure to ozone at 0.15 ppm. 

Charriere-Ladreix, Y. and Tissut, M., Foliar flavonoid distribution during Spinacia chloroplast isolation, Planta, 151, 309, 1981. 

Burke, J.J., R.F. Wilson, and J.R. Swafford (1982). Characterization of chloroplasts isolated from triazine-susceptible and triazine-resistant biotypes of Brassica campestris L. Plant Physiol., 70 24—29. 

Oettmeier, W., K. Masson, C. Fedtke, J. Konze, and R.R. Schmidt (1992). Effect of different photosystem II inhibitors on chloroplasts isolated from species either susceptible or resistant toward s-triazine herbicides. Pestic. Biochem. Physiol., 18 357-367. 

So far, only one in vitro system for ABA biosynthesis has been described (44). In this study chloroplasts isolated from ripening avocado fruits incorporated mevalonate into ABA. Upon lysing the cell-free system in dilute buffer, incorporation of label into ABA increased considerably, indicating that the chloroplast membrane was a major barrier to the penetration of mevalonate. Although these preliminary results demonstrate that ABA can be synthesized within the chloroplast, the possibility that synthesis can also take place outside the chloroplasts, can by no means be ruled out. 

B. Suppose that chloroplasts isolated from such a cell have the same osmotic responses as in Problem 2.5. What is the volume of such chloroplasts in vivo Assume that activity coefficients are 1 and that the temperature is 20°C. 

Imhoff, V, and Bourdu, R., 1973,. Formation, d inositol par les chloroplasts isoles de pois. Phytochemistry 12 331-336. 

Alkaloid metabolism in lupine was proved by Wink and Hartmann to be associated with chloroplasts (34). A series of enzymes involved in the biosynthesis of lupine alkaloids were localized in chloroplasts isolated from leaves of Lupinus polyphylls and seedlings of L. albus by differential centrifugation. They proposed a pathway for the biosynthesis of lupanine via conversion of exogenous 17-oxosparteine to lupanine with intact chloroplasts. The biosynthetic pathway of lupinine was also studied by Wink and Hartmann (35). Two enzymes involved in the biosynthesis of alkaloids, namely, lysine decarboxylase and 17-oxosparteine synthetase, were found in the chloroplast stoma. The activities of the two enzymes were as low as one-thousandth that of diaminopimelate decarboxylase, an enzyme involved in the biosynthetic pathway from lysine to diaminopimelate. It was suggested that these differences are not caused by substrate availability (e,g., lysine concentration) as a critical factor in the synthesis of alkaloids. Feedback inhibition would play a major role in the regulation of amino acid biosynthesis but not in the control of alkaloid formation. 

Generation of Superoxide Radicals and Hydrogen Peroxide in Chloroplasts. Isolated, illuminated chloroplast thylakoids slowly take up oxygen in the absence of added electron acceptors. This phenomenon was first observed by Mehler (7) and is often known as the "Mehler reaction. The reaction appears to result from the reduction of O2 to the... 

In an effort to generate images of the morphology of thylakoid membranes inside plant chloroplasts, isolated chloroplasts resuspended in a buffer solution containing a herbicide, 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), have often been used [10, 11]. The DCMU inhibits photosynthesis by blocking electron transport at the electron acceptor side of PSII and enhances chlorophyll fluorescence from PSII. 

The activity of some enzymes of phosphorus metabolism in chloroplasts isolated in a nonaqueous medium 4837. 

Ongun, A., W.W. Thomson, and J.B. Mudd Lipid composition of chloroplasts isolated by aqueous and nonaqueous techniques J. Lipid Res. 9 (1968) 409 15. 

Park RB, Kelly J, Drury S et al. The Hill reaction of chloroplasts isolated from glutaraldehyde-fixed spinach leaves. Proc Natl Acad Sd USA 1966 55 1056-62. 

The effects of cadmium and lead (Pb) has been assessed in chloroplasts isolated from lucerne (Medicago sativa). The presence of Cd and Pb led to the inhibition of the PSII activity. The inhibition appeared to be located on the oxidizing side of PSII because the addition of artificial donors restored the electron flow. 

Chloroplast Isolation. Chloroplasts of peas, spinach (Spin-acia oleracea L.), and biotypes of Amaranthus hybridus L. suscep-t1b1e or resistant to 2-triazines were isolated and stroma-free thylakoids prepared as previously described (17). Intact chloroplasts were obtained from pea leaves following the method of Blair and Ellis (18). 

One additional line of evidence that independently implicates a 32 kDal polypeptide in triazine binding comes from studies on chloroplasts isolated from a maize mutant that specifically lacks the stainable 32 kDal polypeptide and the rapidly labeled 34 kDal polypeptide. Chloroplasts of this PS Il-less lethal mutant lack binding sites for radioactive atrazine (35). 

Many experiments into the secondary effects of photosynthetic inhibitor herbicides have been performed with isolated chloroplasts, Isolated chloroplasts provide a convenient system for studying the generation and quenching of singlet oxygen. 

We compared the membrane lipid composition of chloroplasts isolated from species of common groundsel (Senecio vulgaris L.), lambsquarters (Chenopodium album L.), and pigweed (Amaranthus hybridus L.) (Table VIII) (W. 

A greater proportion of the total lipid from chloroplasts isolated from resistant species was found in the polar (membrane) lipid fraction. When this fraction was separated into individual lipid classes, chloroplasts from resistant biotypes contained higher proportions of MGDG and PE,and lower proportions of DGDG and PC than chloroplasts from susceptible species. There were no consistent trends for other classes of phospholipids. The proportional differences are a reflection of qualitative differences. However, a ratio of the resistant to susceptible value of greater than 1 in Table VIII indicates a quantitative increase in that lipid class and vice versa. 

Chloroplasts isolated from spinach (Spinacia oleracea) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of a- and /3-amylase and /3-D-glucosidase ( maltase ) (see p. 455). 

It is evident that the fiiU range of amino acids must be synthesized in, or gain entry to, chloroplasts to facilitate protein synthesis (Ellis, 1977). The apparent concentration of several amino acids in isolated chloroplasts would be sufficient to influence the activity of regulatory enzymes associated with amino acid biosynthesis (Miflin, 1976 Chapman and Leech, 1979). Furthermore, the synthesis of aspartate from [ K] ]02 was 40-60 times greater than the synthesis of threonine or lysine in Vida faba chloroplasts (Kirk and Leech, 1972). These results would be consistent with functional control of amino acid synthesis by endogenous end-products. However, the amounts of important end-product effectors, such as lysine, threonine, and isoleucine, detected in chloroplasts isolated firom different plant species, (Aach and Heber, 1967) or from tissues of different developmental stages (Chapman and... 

Heinz, E Roughan, PG. Similarities and differences in lipid metabolism of chloroplasts isolated from 18 3 and 16 3 plants. Plant Physiol, 1983, 72, 273-279. 

Metabollsme des composes phenollques chez le Petunia V. utilisation de la phenylalanine par des chloroplastes isoles, PI. Scl. Letters lo, 225-234 (1977)... 

Seed was obtained from a triazine-resistant and a triazine-susceptible population of smooth pigweed ( Amaranthus hybridus ) generously gifted by Drs. D. Ort and R.A. Liebl. The growing condition of the plants and the chloroplast isolation are described in [1]. 

Fig. 3 Relationship between qE and pH for chloroplasts isolated from dark-adapted spinach leaves wi th negligible levels of zeaxanthin (open circles) and from pre-illuminated leaves (30 mins at 1000 Umol.mT, s ) with high zeaxanthin levels (closed circles). Chlorophyll...

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). 

Chloroplasts isolated from both vegetative and reproductive fronds exhibited similar trends in PS to varying PPFD and chlorophyll content within the electrode vessel. Typical light saturation curves are J.llujtrated in Figure 1. Maximum net PS occurred a1 60 Mmol m s (PPFD) for a j lorcphyll content of 25 Mg cm as opposed to 250 Mmol s (PPFD) for a... 

The experiments were performed with suspensions of envelope-free chloroplasts isolated from spinach at 20 C temperature. The initial pH was 8.0 and did never deviate more than 0.05 units. The intensity of the actinic light was 300 Wm-2 unless otherwise stated. Changes of external pH (by glass electrode measurement) and of fluorescence (from the added indicator of Internal pH) were monitored simultaneously. Changes of the ATP concentration were obtained from the change of external pH (0.94 H consumed per ATP formed). Light-induced electron flow (in the presence of ferricyanide as electron acceptor) was obtained from the decrease of external pH (1 H liberated per electron transported). Transmembrane ApH was obtained from the fluorescence quenching of N-(l-naphtyl)ethylenediamine (NED) or from the external pH jump after a pulse of 50 pM imidazole. The H/e determination was performed as described in [1]. 

The experiments were performed with envelope-free chloroplasts isolated from spinach. 

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