Isolation of Mesophyll Cells and Bundle Sheath Cells from <i>Digitaria sanguinalis</i> (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology

Edwards, Gerald E.; Black, Clanton C.

doi:10.1104/pp.47.1.149

Cited by 71 publications

(28 citation statements)

References 24 publications

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“…A new procedure for purification of M and BS chloroplasts was developed based on suggestions from A. Barkan and R. Bassi and on published protocols (Edwards and Black, 1971;Kannangara et al, 1977).…”

Section: Methodsmentioning

confidence: 99%

“…The top 4-cm sections of the 2nd and 3rd leaves were harvested ;2 h after the onset of the light period. We used a mechanical procedure for purification of M and BS chloroplasts, following suggestions from A. Barkan and R. Bassi and protocols adapted from Edwards and Black (1971) and Kannangara et al (1977). This procedure is based on the much higher resistance of the BS cell wall to mechanical grinding as compared with the M cell wall.…”

Section: Purification Of Bs and M Chloroplasts From Leaf Tips Of Younmentioning

confidence: 99%

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Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

et al. 2005

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Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway.

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Section: Methodsmentioning

confidence: 99%

Section: Purification Of Bs and M Chloroplasts From Leaf Tips Of Younmentioning

confidence: 99%

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

et al. 2005

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“…Bundle sheath cells also may fix some atmospheric CO2 (about 15% of the total CO2 fixed by the leaf) via the regular pentose cycle (14).…”

Section: Methodsmentioning

confidence: 99%

“…Recently we developed a method for separating the two cell types (14,15), and have undertaken an investigation of the properties of each cell type. This paper is a report on some spectral and physical measurements, Hill reactions, and enzyme activities of the photosynthetic apparatus of these two distinct cell types isolated from Digitaria sangiuinalis leaves.…”

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confidence: 99%

Spectral, Physical, and Electron Transport Activities in the Photosynthetic Apparatus of Mesophyll Cells and Bundle Sheath Cells of Digitaria sanguinalis (L.) Scop.

1971

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Isolated mesophyll cells and bundle sheath cells of Digitaria sanguinalis were used to study the light-absorbing pigments and electron transport reactions of a plant which possesses the C4-dicarboxylic acid cycle of photosynthesis. Absorption spectra and chlorophyll determinations are presented showing that mesophyll cells have a chlorophyll a-b ratio of about 3.0 and bundle sheath cells have a chlorophyll a-b ratio of about 4.5.The absorption spectrum of bundle sheath cells has a greater absorption in the 700 nm region at liquid nitrogen temperature, and there is a relatively greater amount of a pigment absorbing at 670 nm in the bundle sheath cells compared to the mesophyll cells. Fluorescence emission spectra, at liquid nitrogen temperature, of mesophyll cells have a fluorescence 730 nm-685 nm ratio of about 0.82 and bundle sheath cells have a ratio of about 2.84. The reversible light-induced absorption change in the region of Po0o absorption is similar in both cell types but bundle sheath cells exhibit about twice as much total P700 change as mesophyll cells on a total chlorophyll basis. The delayed light emission of bundle sheath cells is about one-half that of mesophyll cells. Both mesophyll cells and bundle sheath cells evolve oxygen in the presence of Hill oxidants with the mesophyll cells exhibiting about twice the activity of bundle sheath cells, and both activities are inhibited by 1 jIM 3-(3,4-dichlorophenyl)-1 , l-dimethylurea. Ferredoxin nicotinamide adenine dinucleotide phosphate reductase is present in both cells although it is about 3-or 4-fold higher in mesophyll cells than in bundle sheath cells. Glyceraldehyde 3-P dehydrogenases, both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, are equally distributed in the two cell types on a chlorophyll basis. Malic enzyme is localized in the bundle sheath cells.We interpret the data as evidence for the presence of a complete chloroplast electron transport system from oxygen evolution to pyridine nucleotide reduction in both mesophyll and bundle sheath cells. However, there is a quantitative difference in the distribution of photosystem I and photosystem II components in the two photosynthetic cells with about a 3-fold higher photosystem I-II ratio in the bundle sheath cells than in the mesophyll cells. A scheme is proposed to accommo-

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“…Both these methods, enzymatic or mechanical, of cell isolation are not mutually exclusive in preparing leaf cells as no single method is applicable to all plant forms (5,6,13,17 …”

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confidence: 99%

Physiological Studies with Isolated Leaf Cells

Kulandaivelu

Gnanam²

1974

Plant Physiol.

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MATERIALS AND METHODSPlant Materials. The plant materials used in this study were cultivated in the University botanical garden, Madurai. Fresh and fully developed green leaves were harvested and used immediately, or stored at 1 to 5 C for 1 to 2 hr before experimentation. Euglena gracilis Z, was grown autotrophically at 27 C in a modified Hutner medium (15). Chlorella vulgaris was grown in a 3-liter vessel containing 2 liters of inorganic medium (10). The cells were harvested after 6 to 9 days, washed twice with 0.01 M NaCl, and suspended in 0.05 M phosphate buffer pH 7.0.Isolation of Mesophyll Cells. Mesophyll cells were isolated and used following the procedure of Gnanam and Kulandaivelu (9). Chlorophyll content was determined by the method of Arnon (1).Absorption Spectrum. The absorption spectrum of each cell suspension was measured in a Beckman DK-2A ratio recording spectrophotometer, with opal glass placed between the slit and the sample tube for correcting the light scattering as described by Takebe et al. (19). Incorporation of Labeled Compounds by Mesophyli andAlgal Cells. "4CO2 fixation by mesophyll cells was carried out with modification of the method described by Bassham and Calvin (2). The reaction mixture (30 ml) contained 50 mm potassium phosphate buffer, pH 7.2, 35 mm NaCl, 5 mm MgCl,, 20,FCi NaH`4C0, (15 mCi/mmole), and S X 107 cells. Light was supplied by a 500 w projector-reflector lamp focused on the reaction vessel. The temperature of the reaction mixture was maintained at 20 + 1 C. The reaction mixture was stirred constantly by bubbling compressed sterile air. To obtain a steady state of CO2 fixation, cells were preilluminated for 10 min before the addition of labeled compounds.Amino acid incoporation by leaf cells was studied in a reaction mixture of 6 ml containing 50 mm tris-HCl buffer, pH 7.5, 20 mm NaCl, 10 mm MgCl2, 3 uCi of L-14C leucine (29.4 mCi/mmole), and 1.2 X 107 cells. Temperature of the reaction mixture was maintained at 25 C. Aliquots of 1 ml were pipetted at regular intervals into tubes containing 0.2 ml of 20% trichloroacetic acid and centrifuged at 3000g for 5 min. The pellet was washed twice with 5 mm 'C-leucine and suspended in 0.1 M tris-HCl buffer, pH 7.5. Cells were homogenized and the protein was precipitated by 5% trichloroacetic acid and redissolved in 0.01 N NaOH for radioactive counting.Chromatography and Radioautography. Two-dimensional paper chromatography and radioautography were carried out according to the method described by Bassham and Calvin (2). The individual compounds were identified on the paper by cochromatography with authentic compounds except for phosphoglycolate. The position of P-glycolate is marked tentatively by its relative position in the chromatograms developed using the same solvent systems by others (4

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Isolation of Mesophyll Cells and Bundle Sheath Cells from Digitaria sanguinalis (L.) Scop. Leaves and a Scanning Microscopy Study of the Internal Leaf Cell Morphology

Cited by 71 publications

References 24 publications

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

Spectral, Physical, and Electron Transport Activities in the Photosynthetic Apparatus of Mesophyll Cells and Bundle Sheath Cells of Digitaria sanguinalis (L.) Scop.

Physiological Studies with Isolated Leaf Cells

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