Translocation and Metabolic Conversion of <sup>14</sup>C-Labeled Assimilates in Detached and Attached Leaves of <i>Phaseolus vulgaris</i> L. in Different Phases of Leaf Expansion

Exposure of white bean seedlings to phytotoxic burdens of Co, Ni, or Zn reduced the export of "C-photoassimilates from the nearly fully expanded unifoliate leaves. Little "C reached the major sink areas, the young trifoliate leaves and the root tips, of seedngs exposed to metal. The unifoliate leaves accumulated sucrose, reducing sugars, and starch. These effects were evident within 1 or 2 days.The most general symptoms of metal toxicity in plants are stunting and chlorosis (7). Yield reductions could occur by excess metal directly or indirectly inhibiting either assimilate production in source leaves, translocation from source to sink, utilization in sink regions, or several of these. Various metal ions have adverse effects on respiration and photosynthesis in intact organisms, ranging from algae and lichens to vascular plants, as well as on isolated mitochondria and chloroplasts (5,10,16,19,22). In this context, it is particularly noteworthy that carbohydrates accumulate in shoots of barley (1) and starch accumulates in leaves of white beans (23) on exposure of seedlings to excess Co, Ni, or Zn. Agarwala et aL (1) suggested that the carbohydrate accumulations were due to metal interferences with carbohydrate metabolism. Rauser (23) gave the alternative interpretation that starch accumulations reflected reduced translocation out of leaves which normally export carbon.The aim of this work was to determine the levels of sucrose and reducing sugars in relation to starch in seedlings of Phaseolus vulgaris exposed to individual phytotoxic burdens of Co, Ni, and Zn. In addition, we assessed the rate of export of '4C-photoassimilates from unifoliate leaves and their pattern of distribution in bean seedlings exposed to excess metal. Sucrose, Reducing Sugars, and Starch Contents. Unifoliate leaves were harvested between 1200 and 1300 h and quickly dropped into 40 ml boiling 13.7 mol 1-1 ethanol in water and refluxed for 30 min. After reextraction with 40-and 20-ml portions of aqueous ethanol, the leaves were completely decolorized. The three alcohol extracts of each leaf were combined and evaporated nearly to dryness by gently warming. The residue was taken up in 20 to 30 ml deionized H20 filtered quantitatively through a glass fiber filter, and the filtrate made to a total volume of 50.0 ml with water. A 10.0-ml subsample of the filtrate was pipetted into a 15-ml centrifuge tube containing 1 ml each of swollen Dowex l-X8 anion exchange resin (200-400 mesh, C1-form) and of Dowex 50-W cation exchange resin (100-200 mesh, H+ form). The contents were mixed thoroughly and centrifuged at l,500g for 2 to 3 min. The supernatant was decanted and the resin mixture was extracted once with 4 ml water. Both supernatants were combined and made to a total volume of 15.0 ml. MATERIALS AND METHODSVariable volumes of the resin-treated supernatants were used to determine reducing sugars (18). Other subsamples were digested with 11 units of invertase Sigma Chemical Co.) for 2 h at 50 C. The volumes of supernatant used we...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Carbohydrate Levels and Photoassimilate Export from Leaves of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Samarakoon¹,

Rauser²

1979

“…Numerous workers have postulated that an active process in the minor vein phloem serves to drive mass flow of solutes (1,2,4,5,7,10,14,22,25,26). In support, various investigators have used autoradiography to demonstrate loading of minor vein phloem (9,14,16,17,23). Barrier and Loomis (1) used the term "loading" for the process and proposed that it is an essential step in the translocation process.…”

mentioning

confidence: 99%

Solute Distribution in Sugar Beet Leaves in Relation to Phloem Loading and Translocation

Geiger¹,

Giaquinta²,

Sovonick³

et al. 1973

144

The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze-substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.The sharp concentration increase at the membrane of the sieve element-companion cell complex of the source leaf indicates active phloem loading from the free space at this site. Active loading of the phloem is presumably needed to move the sugar from the chloroplasts of the mesophyll to the sieve tubes against the concentration gradient. The osmotic pressure of the mature sieve element-companion cell complex appears to be approximately the same in source leaf, path, and sink leaf tissue. There is a distinct difference in concentration between the mature sieve element-companion cell complex in the sink and the surrounding mesophyll. The solute distribution suggests that sugar is actively accumulated from the free space by the developing sink leaf tissue.The osmotic values observed in the various cells are consistent with the operation of a mass flow mechanism of translocation driven by active phloem loading and by active accumulation of sugar by sink tissues.

“…Even in studies with intact leaves, only a small fraction of "C assimilate was found to be incorporated into the trichloroacetic acid-insoluble fractions and the synthesis of cellular materials was largely dependent on the external supply from other parts of the plant ("import"). This is true of leaves of all ages (12). That primary products of photosynthesis are predominantly meant for "export" was further evidenced by the fact that over 60% of "C fixed during photosynthesis occurred as free sugar and sugar phosphates.…”

Section: Discussionmentioning

confidence: 87%

Physiological Studies with Isolated Leaf Cells

Kulandaivelu

Gnanam²

1974

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