Chlorophyll Biosynthesis in a Cell-free System from Higher Plants

Rebeiz, Constantin A.; Castelfranco, Paul A.

doi:10.1104/pp.47.1.33

Cited by 62 publications

(25 citation statements)

References 28 publications

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“…These organelles appear to possess certain amounts of RNA and DNA as well as the machinery for the biosynthesis of lipid, protein, and nucleic acids (9). They seem capable of dividing in vitro (17) and of forming their own protochlorophyll and chlorophyll from simple substrates (14,15,22). The differentiation of etioplasts into chloroplasts during greening is accompanied by substantial synthesis and accumulation of chlorophyll (11), carotenoids (12, 13), insoluble proteins, and colorless lipids (10).…”

Section: Introductionmentioning

confidence: 99%

“…61801. 15). Although such a system is presently unavailable, we have found that plastid preparations capable of partial differentiation in vitro can yield useful information.…”

mentioning

confidence: 99%

“…An assessment of cellular control during chloroplast differentiation, growth, maintenance, and division is therefore indispensable in evaluating the degree of autonomy of this organelle. (14,15). Cotyledons to be preirradiated were harvested with full hypocotyl hook as previously described (7).…”

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confidence: 99%

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Chloroplast Maintenance and Partial Differentiation in Vitro

Rebeiz¹,

Larson²,

Weier³

et al. 1973

Plant Physiol.

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Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These (1,6,8,9). These organelles appear to possess certain amounts of RNA and DNA as well as the machinery for the biosynthesis of lipid, protein, and nucleic acids (9). They seem capable of dividing in vitro (17) and of forming their own protochlorophyll and chlorophyll from simple substrates (14,15,22). The differentiation of etioplasts into chloroplasts during greening is accompanied by substantial synthesis and accumulation of chlorophyll (11), carotenoids (12, 13), insoluble proteins, and colorless lipids (10). Likewise, dividing chloroplasts must grow before reaching full size (17). An assessment of cellular control during chloroplast differentiation, growth, maintenance, and division is therefore indispensable in evaluating the degree of autonomy of this organelle. (14,15). Cotyledons to be preirradiated were harvested with full hypocotyl hook as previously described (7). They were placed in beakers with enough distilled H20 to keep them moist and were illuminated with 240 ft-c of white fluorescent light at 28 C for 2.5 or 4.5 hr (15).Chemicals. The commercial sources of chemicals were reported elsewhere (14, 15). Preparation of Unfortified and Fortified Crude Homogenates. "Unfortified" crude homogenates were prepared from 4.5-day-old cotyledons as follows. Four grams of etiolated or greening cotyledons were gently ground with mortar and pestle without sand in 6.0 ml of 0.5 M sucrose and 0.2 M tris-HCl, pH 8.0, at 0 to 5 C. The brei was filtered through four layers of cheesecloth. About 5 ml of unfortified crude homogenates were obtained. "Fortified" crude homogenates were prepared by grinding 4 g of etiolated or greening cotyledons in 6.0 ml of fortified tris-HCl and 0.

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Section: Introductionmentioning

confidence: 99%

“…61801. 15). Although such a system is presently unavailable, we have found that plastid preparations capable of partial differentiation in vitro can yield useful information.…”

mentioning

confidence: 99%

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Chloroplast Maintenance and Partial Differentiation in Vitro

Rebeiz¹,

Larson²,

Weier³

et al. 1973

Plant Physiol.

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“…Cotyledons were harvested with full hypocotyl hook as previously described (2). They were placed in beakers with enough distilled H20 to keep them moist and were irradiated with white fluorescent light for 4 hr (8). De-veloping chloroplasts were isolated from the greening cotyledonary tissues.…”

Section: Methodsmentioning

confidence: 99%

“…Alpha Green) were germinated in vermiculite (Terra Lite) at 24 C in complete darkness for 4 days (7,8). Cotyledons were harvested with full hypocotyl hook as previously described (2).…”

Section: Methodsmentioning

confidence: 99%

The Biosynthesis of Metal Porphyrins by Subchloroplastic Fractions

1972

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The biosynthesis of Mg protoporphyrin monoester, protochlorophyll and chlorophyll a and b by crude homogenates, isolated etioplasts, and differentiating chloroplasts has recently been described by . In the present communication we wish to report briefly on the capacity of subchloroplastic fractions to form free porphyrins and metal porphyrins. MATERIALS AND METHODSCucumber seeds (Cucumis sativus L. cv. Alpha Green) were germinated in vermiculite (Terra Lite) at 24 C in complete darkness for 4 days (7,8). Cotyledons were harvested with full hypocotyl hook as previously described (2). They were placed in beakers with enough distilled H20 to keep them moist and were irradiated with white fluorescent light for 4 hr (8). De The reaction was stopped by the addition of 15.0 ml of acetone:0.1 N NH4OH (9:1 v/v) and centrifuged at 39,000g for 10 min. The pellet was saved for the extraction of free porphyrins and the acetone: NH4OH supernatant containing metal porphyrins and free porphyrins was extracted with an equal volume of hexane. The hexane extracted most of the ,8-carotene and chlorophyll. The metal porphyrins remaining in the hexane-extracted acetone: NH4OH supernatant were transferred to ether as previously described (6). The proteinaceous pellet and the aqueous acetone phase that was left after metal porphyrins extraction were pooled and used for the extraction of free porphyrins as described previously (6).Free porphyrins were separated by ascending paper chromatography on Whatman No. 3 MM paper in 2,6-lutidine:0.05 N NH4OH (5:3.5 v/v). Metal porphyrins, in ether, were separated on thin layers of Silica Gel H in benzene-ethyl acetateethanol (8:2:2 v/v). All fractions were routinely monitored by their absorption spectra and recorded with a Beckman double beam spectrophotometer Model DK-2A. All absorbancies used in quantitative determinations were derived from the appropriate spectra.The amount of uroporphyrin in acidified, saturated sodium acetate (pH 2.5) was estimated from absorbancy at 402 nm using the molar extinction coefficient (5.41 X 106) and the correction factors reported by Rimington and Sveinsson (10) for uroporphyrin in 0.5 N HCI.The copro + protoporphyrin fraction in ether containing mostly coproporphyrin was estimated from its absorbancy at 401 nm and a molar extinction coefficient of 1.8 X 105 (6). When protoporphyrin was predominant in this fraction, its absorbancy at 404 nm and a molar extinction coefficient of 1.5 X 105 were used instead (6).

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