Metabolism of Separated Leaf Cells

Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of "C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl2. Unloading was stimulated by 15 millimolar ethyleneglycol-bis(6-aminoethyl ether)-N,N,N',N'-tetraacetic acid which partially reversed the inhibitory effects of Ca21. Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses.

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confidence: 47%

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confidence: 99%

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue

Porter¹,

Knievel²,

Shannon³

1985

“…Present plant cell isolation procedures (6,12,19,21) are not entirely satisfactory in that they require a high osmotic concentration, and 1 to 3 hr are required for high cell yields. A method is described here for the rapid isolation, in high yield, of photosynthetically active mesophyll cells from soybean in the presence of a physiological concentration of osmoticum.…”

Section: Introductionmentioning

confidence: 99%

Rapid Isolation of Mesophyll Cells from Leaves of Soybean for Photosynthetic Studies

Servaites

Ogren

1977

Mesophyll cells were rapidly isolated from soybean (Glycine max IL.]) leaves using a combined Macerse enzyme-stirring technique.About 50% to 70% of the leaf cells on a chlorophyll basis from 3 grams of leaves could be isolated in 15 minutes. The cells obtained by this method were capable of high rates of photosynthesis even after storage in the dark for periods of up to 9 hours. The C02-saturated rate of photosynthesis increased from 5 ,um COJmg Chi hour at 5 C to 170 Am COimg Chl-hour at 40 C. At atmospheric CO2 concentration, the rate varied from 5 to 55 Fm COJmg Chi hour over this temperature range. The reduced temperature response of photosynthesis at low CO2 concentration was due to an increased Km(CO2) of the cells with increasing temperature. The products of photosynthesis in the isolated cells were similar to the products of leaf photosynthesis.Using enzyme digestion techniques, leaf mesophyll cells and protoplasts have been isolated from a number of C3 and C4 plant species (11-13, 19, 21). Isolated cells and protoplasts appear to be ideal material for studying photosynthesis, photorespiration, and their regulation, for unlike chloroplasts and algae, leaf mesophyll cells from C3 plants contain the complete pathway of photorespiration as well as the photosynthetic cycle.Present plant cell isolation procedures (6,12,19,21) are not entirely satisfactory in that they require a high osmotic concentration, and 1 to 3 hr are required for high cell yields. A method is described here for the rapid isolation, in high yield, of photosynthetically active mesophyll cells from soybean in the presence of a physiological concentration of osmoticum.Photosynthesis in these cells has been characterized to determine the similarity to leaf photosynthesis and the usefulness of these preparations for studying the kinetics and biochemistry of photosynthesis and photorespiration. with three times the amount of iron (Sequestrene 3302). Plants were grown at 35 klux in a chamber with a 16-hr photoperiod (30 C light/20 C dark) for 3 weeks, and then transferred to a chamber having a 6-hr photoperiod. After 1 week of short day treatment, a mature leaf, usually from the third to fifth emergent trifoliate, was shaded with aluminum foil for 1 to 2 days. On the day of cell isolation, the leaf was illuminated for 1 hr and detached from the plant. At the time of leaf removal, leaf area was about 0.6 dM2, specific leaf weight was 1.7 to 2 g fresh wt/dm2, and the leaf contained 3.5 to 4 mg Chl/g fresh wt.Cell Isolation. The detached leaf was rinsed with distilled H20 and the midrib was removed. The leaf was placed in a Petri dish containing 20 ml of infiltration medium consisting of 20 mM MES (pH 5.8), 12.5 mm K2SO4,2% (w/v) PVP-40, and 3% (w/v) Macerase (Calbiochem), and cut into strips (1 mm x 1 cm). The Macerase was purified prior to use by (NH4)2SO4 precipitation and passage through Sephadex G-25. The leaf strips were vacuum-infiltrated for 15 sec (12), and the infiltration medium discarded. The leaf strips and a stirring bar were...

“…Each flask also contained 0.1 ml of the radioactive substrate making a final volume of 2.6 ml. This' method of cell isolation was a modification of previously described methods (7,10,17) and identical to that previously used (1).The assay methods for the processes studied were also essentially the same as previously described (1). After the appropriate radioactive substrate was added to the cell preparation, the flasks were stopped and placed in a 25 C shaking water bath illuminated from above with fluorescent lamps with an intensity of 4,300 lux at the level of the flasks.…”

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confidence: 99%

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confidence: 99%

Metabolic Activity of Isolated Leaf Cells of Phaseolus vulgaris in Relation to Leaf Development

Devilliers

Ashton²

1977

Mesophyil cells were isolted from primary leaves of 5-to 21-day Phaseolus vulgaris plants. The rate of photosynthesis and respiration, and RNA, protein, and lipid synthesis was determined for these cels. Appropriate 14C substrates and product purification procedures were used for each process prior to liquid scintillation counting. The size of the leaves increased about 5-fold between days 5 and 11, and then remained relatively constant. The greatest increase in size occurred between days 5 and 6. The age of the leaf from which the cells were isolated had a pronounced effect on the rate of anl of these processes. The largest chages occurred during the period of leaf expansion (days 5-11).Initially the rate of RNA, protein, and lipid synthesis increased rapidly, maintained a muximum rate for only 1 day (day 6 or day 7), and then dedined. The rate of photosynthesis increased more slowly reaching a amum at day 9, remained relatively constant until day 15, and then dedined. The rate of respiration decreased during the first 4 days to low level which was maintained throughout the experiment. The time course patterns of these biochemical processes in isolated cells were similar to those which have been reported for intact leaves. It seems that isolation of leaf cells does not modify their metabolic activity.During the past several years, techniques have been developed for the isolation of separated mesophyll cells from higher plants (7,10,12, 20,21). Isolation of metabolically active cells constitutes a useful experimental system for biochemical studies of cellular processes in plants. This technique is also useful in helping to bridge the gap between those results obtained with isolated enzyme systems and excised tissue or intact plant systems. It has been shown that these cells are capable of performing metabolic processes like photosynthesis (1, 10), respiration(1), RNA and protein synthesis (1, 7), and lipid synthesis (1).Changes in the level of metabolic activity of leaves during their development, maturation, and senescence have been observed. These include changes in Chl content (2, 19), protein content (8), photosynthesis (2-6, 14, 18), respiration (5), RNA synthesis (16), and protein synthesis (6). These studies have been conducted on intact leaves; however, similar studies on isolated single cells have not been performed. During recent investigations on the mechanism of herbicide action using isolated leaf cells (1), we found that age of the leaf from which the cells were isolated had considerable influence on the reaction rate of several processes. These observations prompted this systematic investigation on the relation of leaf age to the rate of photosynthesis, respiration, RNA synthesis, protein synthesis, and lipid synthesis in isolated leaf cells. Primary leaves were excised 4 to 5 hr after initiation of the light period at 5, 6, 7, 9, 11, 13, 15, 17, and 21 days. Four of these leaves were used to determine the stage of leaf growth at the various harvest times and the remainder used for the ce...