Photosynthetic Studies With Leaf Cell Suspensions From Higher Plants

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO2 fxation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH'4CO3, and the kinetics of 14C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO 2fxation is similar in the different mesophyll tissues from which these cells were derived.Compartmentation of photosynthetic processes between the mesophyll and bundle sheath cells of C4 plants has been partially elucidated (6,15 The kinetic data of 14C incorporation into glycine and serine were markedly different in extracts of shavings taken from the upper ("palisade") and lower ("spongy") surfaces of frozen coltsfoot leaf which had been exposed to '4C02 before freezing (22). Outlaw and Fisher (25) found that the percentage of '4C in ths combined glycine and serine fraction from extracts of microtome sections of palisade parenchyma after pulse-labeling a Vicia faba leaf was smaller than in sections of spongy parenchyma. Moreover, differences in the internal organization of microbodies from the different mesophyll cell types have been observed (9). These observations suggest the possibility of differences in photorespiration. We have isolated palisade parenchyma cells and spongy parenchyma cells from V. faba leaflet, and determined levels of certain enzyme activities involved in photosynthetic carbon metabolism and the kinetics of photosynthetic '4C incorporation into various compounds. MATERIALS AND METHODSPlant Material. V. faba L. plants were grown on Hoagland solution in a soil-vermiculite mixture in a growth cabinet, set on a 14-hr day at 21 C and 15 C at night. Irradiance from incandescent and fluorescent bulbs was 4000 ft-c at plant level.Cell Preparation. Young, fully expanded leaves were used from 3-to 5-week-old plants. About 2.5 g of leaflet, from which the lower epidermis had been stripped, were placed in a 125-mi side arm flask and vacuum-infiltrated (300 mm Hg, minimum pressure) with 20 ml of maceration medium. The medium was similar to that used by others (7, 17, 32) and contained 0.8 M sorbitol, 20 mm K2SO4, 1 mm KNO3, 0.2 mm KH2PO4, 0.2 mM MgSO4, 1 mM CaC12, 1 ,M KI, 0.01 MM CuSO4, 300 ug/ml Cephaloradine (E. Lilly). The medium was adjusted to pH 5.8 before adding 1% (w/v) Macerase (Calbiochem). The stripped leaflets were incubated at 30 C in this solution on a shak...

Section: Methodsmentioning

confidence: 97%

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L.

Outlaw

Schmuck²,

Tolbert³

1976

“…Mechanical techniques for homogenizing leaves to isolate mesophyll cells also have been reported (1,13,24). Racusen and Aronoff isolated a homogenous mesophyll cell preparation from soybean leaves by homogenizing leaves with a test tube homogenizer, filtering through bolting silk, and separating the cells by low speed centrifugation (24 (4,18) in crude leaf extracts of C4-cycle plants.…”

Section: Resultsmentioning

confidence: 99%

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

Edwards

Black

1971

Slack (15,27) directed the efforts of research workers to correlate leaf anatomy with CO2 fixation cycles (9, 18). Species which utilize the C4-cycle possess chloroplast-containing bundle sheath cells while species which only utilize the pentose cycle of photosynthesis do not.In the early enzyme studies of Hatch and Slack (15, 18, 27) both PEP-carboxylase and RuDP-carboxylase were detected in crude leaf extracts of C4-cycle plants. The level of RuDP-carboxylase was only about one-tenth the rate of leaf photosynthesis; thus the pentose cycle was not given a major role in photosynthetic CO2 fixation in these plants. However, recent data on the level of RuDP-carboxylase indicated that these early studies were in error and the pentose cycle may play a major role in photosynthesis in C4-cycle plants (3,4,10). Since the anatomical studies indicated that two types of photosynthetic cells were present in the same leaf and the biochemical studies indicated that two carboxylases were present, we reasoned that in order to characterize leaf photosynthesis in C4-cycle plants one must examine the metabolism of each cell type. Recently, we presented some biochemical studies with Digitaria sanguinalis on the isolated photosynthetic cell types, demonstrating that each cell type has a characteristic metabolism (10). This manuscript presents a technique for separating mesophyll cells, bundle sheath strands, and bundle sheath cells from fully differentiated leaves of D. sanguinalis. Microscopic evidence for separation is presented and further data on the internal leaf anatomy of C4-cycle plants is presented.Since a large body of literature is available on various aspects of spinach metabolism, particularly photosynthesis, we also developed a technique for isolating mesophyll cells from spinach leaves which we utilize as being typical of cells containing the pentose cycle of photosynthesis. MATERIALS AND METHODS

“…Mesophyll cells were isolated and used following the procedure of Gnanam and Kulandaivelu (9). Chlorophyll content was determined by the method of Arnon (1).…”

Section: Methodsmentioning

confidence: 99%

“…Another approach to the preparation of leaf cells essentially involving mild and careful grinding of leaves in a mortar under isotonic conditions has been reported by Gnanam and Kulandaivelu. (9).…”

mentioning

confidence: 99%

Physiological Studies with Isolated Leaf Cells

Kulandaivelu

Gnanam²

1974

Self Cite

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