Stripping thin layers from chromatographic plates for radiotracer measurements

The rte of phloem loading, its selectivity, and the disposition of labeled carbon were studied following application of 14C-labeled sugars to the free space of source leaves of sugar beet (Beta vulgaris L.). Buffered 10 mm solutions of 14C-labeled sucrose, fructose, stachyose, mannitol, 3-0-methyl glucose or L-glucose were applied to the abraded epidermis of source leaves held in the dark. Distribution of the labeled carbon from sugar taken up from the free space was studied by microdensitometry of autoradiographs. Uptake of labeled sugar from the free space, partition between mesophyll and minor veins, metabolic conversions, export and respiration were followed during the 3-hr time course studies. Rates of sugar uptake into the minor veins, flux rates through the sieve element-companion cell complex membrane and concentration ratios between free space and the interior of the minor vein phloem cells were compared for the six sugars studied for evidence of active uptake. The composition of the free space solution in leaves photosynthesizing in 14CO2 was studied by vacuum infiltration of the source leaf air spaces and removal of the solution by centgation. Labeled compounds in this solution were compared to those in an aqueous ethanol extract of the same leaf pieces.The results in sugar beet source leaves support the concept of direct, active uptake of sucrose from free space into minor veins. This is not the case for fructose, 3-0-methyl glucose, mannitol, or stachyose. The latter two sugars, which are translocated in some plants, are not loaded into the minor veins at a rate sufficient to make them a significant component of the material translocated. The rate of phloem loading is controlled in part by mesophyll metabolism, especially as it affects the availability of sucrose to the free space. Both the rate and selectivity of export are controlled by uptake from the free space into the sieve element-companion cell complex of the minor veins.Phloem loading in leaves of higher plants has an important role in the productivity of crops. Because the bulk of material *that is translocated must first undergo phloem loading, factors which affect this latter process are likely to play a significant part in determining the partitioning of assimilate between export and retention in source leaves. The major portion of material exported from the leaves of higher plants consists of a relatively few sugars and sugar alcohols (20). Data from previous studies (5,6,16) indicate that phloem loading involves active uptake of sugar from the apoplast. An understanding of selectivity and other characteristics of active uptake from the free space of the various exportable sugars seems to be an important starting point for studying the control of export from leaves. 2 Abbreviations: SA: specific activity; se-cc: sieve element-companion cell; 3-0-MG: 3-0-methyl glucose.Mechanisms which affect the availability of various sugars to the free space and their selective uptake into the phloem are likely to control export from the leav...

Section: Methodsmentioning

confidence: 99%

Sugar Selectivity and Other Characteristics of Phloem Loading in Beta vulgaris L.

Fondy¹,

Geiger²

1977

“…Developed plates were autoradiographed and radioactive components extracted according to Redgwell et al. (25).…”

Section: Radioisotope Labelingmentioning

confidence: 99%

“…To determine specific radioactivities, radioactivities were obtained from the two-dimensional TLC sugar and the thinlayer electrophoresis phosphate ester separations as described by Redgwell et al (25). Sugars were quantified by GC/flame ionization detection.…”

Section: Radioisotope Labelingmentioning

confidence: 99%

Mannitol Synthesis in Higher Plants

Loescher¹,

Tyson²,

Everard³

et al. 1992

118

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase 14C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent Km (M6P) was 15.8 millimolar, but the apparent Km (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg2+, Ca2+, Zn2+, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPHdependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.Sugar alcohols (acyclic polyols or alditols) are obtained when the aldo or keto group of a sugar is reduced to a hydroxyl. Mannitol, the most frequently occurring sugar alcohol in plants, is particularly abundant in algae and has been detected in at least 70 higher plant families. It is a major carbohydrate in many members of some dicot families, e.g. the Scrophulariaceae, Oleaceae, Rubiaceae, and Apiaceae (2). Until recently, however, little information has been available on mannitol's role in higher plants (16,17 that it is an early photosynthetic product (27, 29) and present in phloem tissue or phloem exudates of celery (family Apiaceae) (9) and species in many other families, e.g. the Oleaceae (30). Other physiological roles have been proposed, including osmoregulation, storage and recycling of reducing power, and service as a compatible solute (16,17), but very little is known of mannitol metabolism in higher plants. A M6PR2 has been reported as being located in the cytosol of mesophyll protoplasts from celery (27). Preliminary labeling data derived from celery and privet were responsible for the initial assays for reductase activity with M6P and mannitol 1-P as substrates.Here we demonstrate the formation of M6P and mannitol 1-P as early photosynthetic products in celery and privet (family Oleaceae). We also report evidence for the role and importance ofM6PR and its characteristics in mannitol biosynthesis in celery. MATERIALS AND METHODS Plant Mate...

“…The amino acids were separated by two-dimensional TLC using the solvent systems of Haworth and Heathcote (10) and developing the plates twice in the second solvent system. Spots were located by autoradiography then removed by covering the outlined area with stripping mixture as described by Redgwell et al (31). To each of the removed spots in acid-washed tubes 0.5 ml borate buffer was added (6).…”

mentioning

confidence: 99%

Oxygen and Carbon Dioxide Effects on the Pool Size of Some Photosynthetic and Photorespiratory Intermediates in Soybean (Glycine max [L.] Merr.)

Hitz¹,

Stewart²

1980

The levels of ribulose 1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), glycolate, glycine, and serine were measured in soybean leaflets during photosynthesis in atmospheres ranging from 1 to 60% 02 and from 0 to 500 microliters per liter CO2.The RuBP level remained constant as CO2 concentration was decreased in atmospheres containing 20 or 60% 02, but increased as CO2 concentration was decreased in atmospheres containing 1% 02. PGA on RuBP carboxylase has been used to explain the direct inhibition of CO., fixation by 02 (20,26). Although the competitive effect of CO) on the oxygenation reaction and, therefore, on glycolate production is well known at CO2 concentrations near or above 1,000 1l/I (2, 35), the extent of labeling in glycolate pathway intermediates (27, 34), the rate of carbon flux through the pathway (24), and the photorespiration rate (23) Intact leaves apparently import carbon into the Calvin cycle and subsequently into the glycolate pathway during photosynthesis in O2 at low CO2 concentration. The specific radioactivity of evolved CO2 (23) and of some glycolate pathway intermediates (24) remains less than that of the fed "CO2 at air levels of 02 and CO2 concentrations below 400 M1/1. If all photorespiratory CO2 arises from the glycolate pathway, this result is most simply explained by a flux of stored carbohydrate into the Calvin cycle and glycolate pathway during photorespiratory CO2 loss (23). Loss of 14C from sucrose-and starch-containing fractions during illumination of C3 leaves in C02-free 02 has also been shown (21), and the specific activity of CO2 evolved into C02-free air after periods of 14CO2 fixation declines more rapidly than the rate of CO2 evolution (7,17). Chloroplasts in the intact leaf may thus be supplied with a carbon source to maintain the level of Calvin cycle intermediates even at low CO2 concentration.To determine whether or not the loss of photorespiratory CO2 at CO2 concentrations near or below the CO2 compensation point is sufficient to reduce the level of Calvin cycle and glycolate pathway intermediates, we have measured the steady-state photosynthesis pool sizes of RuBP, PGA, glycolate, glycine, and serine. Pool size measurements were made at three 02 concentrations and CO2 concentrations varying from 0 to 500 tlI/l. Net CO2