Biomedical subjects

T L Housley

Publications and source records attributed to T L Housley.

13 recordsLinked to original sources

PubMed1992

Purification and Characterization of Wheat beta(2-->1) Fructan:Fructan Fructosyl Transferase Activity.

Fructans are the major storage carbohydrate in vegetative tissues of wheat (Triticum aestivum L.). Fructan:fructan fructosyl transferase (FFT) catalyzes fructosyl transfer between fructan molecules to elongate the fructan chain. The objective of this research was to isolate this activity in wheat. Wheat (cv Caldwell) plants grown at 25 degrees C for 3 weeks were transferred to 10 degrees C to induce fructan synthesis. From the leaf blades kept at 10 degrees C for 4 days, fructosyl transferase activity was purified using salt precipitation and a series of chromatographic procedures including size exclusion, anion-exchange, and affinity chromatography. The transferase activity was free from invertase and other fructan-metabolizing activities. Fructosyl transferase had a broad pH spectrum with a peak activity at 6.5. The temperature optimum was 30 degrees C. The activity was specific for fructosyl transfer from beta(2-->1)-linked 1-kestose or fructan to sucrose and beta(2-->1) fructosyl transfer to other fructans (1-FFT). Fructosyl transfer from oligofructans to sucrose was most efficient when 1-kestose was used as donor molecule and declined as the degree of polymerization of the donor increased from 3 to 5. 1-FFT catalyzed the in vitro synthesis of inulin tetra- and penta-saccharides from 1-kestose; however, formation of the tetrasaccharide was greatly reduced at high sucrose concentration. 6-Kestose could not act as donor molecule, but could accept a fructosyl moiety from 1-kestose to produce bifurcose and a tetrasaccharide having a beta(2-->1) fructose attached to the terminal fructose of 6-kestose. The role of this FFT activity in the synthesis of fructan in wheat is discussed.

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PubMed1991

Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves.

The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with (14)CO(2) was not the same as that after in vitro labeling with (14)C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose.

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PubMed1990

Fructan metabolism in wheat in alternating warm and cold temperatures.

The objective of this research was to develop a system in which the direction of fructan metabolism could be controlled. Three-week-old wheat seedlings (Triticum aestivum L. cv Caldwell) grown at 25 degrees C were transferred to cold temperature (10 degrees C) to induce fructan synthesis and then were transferred to continuous darkness at 25 degrees C after defoliation and fructan degradation monitored. The total fructan content increased significantly 1 day after transferring from 25 degrees C to 10 degrees C in both leaf blades and the remainder of the shoot tissue, 90% of which was leaf sheath tissue. Leaf sheaths contained higher concentrations of fructan and greater portions of high molecular weight fructan than did leaf blades. Fructan content in leaf sheaths declined rapidly and was gone completely within 48 hours following transfer to 25 degrees C in darkness. In leaf blades the invertase activity fluctuated during cold treatment. The activity of sucrose:sucrose fructosyl transferase increased markedly during cold treatment, while fructan hydrolase activity decreased slightly. In leaf sheaths, however, the activity of invertase decreased rapidly upon transfer to cold temperature and remained low. Trends in sucrose:sucrose fructosyl transferase and hydrolase activity in sheaths were the same as those of leaf blades. Sheath invertase and hydrolase activity increased when plants were transferred back to darkness at 25 degrees C, while sucrose:sucrose fructosyl transferase activity decreased. These results indicate that changing leaf sheath temperature can be utilized to control the direction of fructan metabolism and thus provide a system in which the synthesis or degradation of fructan can be examined.

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PubMed1988

Fructan Content and Synthesis in Leaf Tissues of Festuca arundinacea.

The concentration of fructan in tall fescue (Festuca arundinacea Schreb.) changes during growth and in response to environment. The objective of this research was to compare the fructan concentration and fructosyl-transferase activity of tall fescue leaf tissues. Expanding leaves, inner and outer sheaths, and expanded blades of greenhouse-grown tall fescue plants were assayed for fructan concentration and fructosyl-transferase activity. Leaf sheaths contained significantly more nonstructural carbohydrate than did the expanded blade. Sheaths also contained a greater percentage of fructan with more than six sugar residues (long chain fructan), than either the expanded blade or expanding leaf. Expanding leaves contained a greater concentration of fructose and oligosaccharides than did sheath or blade tissues. Expanding leaves also had the greatest fructosyl-transferase activity measured either as radiolabel incorporated into fructans in tissue pieces or protein extracts. Activity of fructosyl-transferase was greater in expanding leaf tissue than in sheath tissues.

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PubMed1987

Fructan Content and Fructosyltransferase Activity during Wheat Seed Growth.

The objective of this research was to determine the changes in fructan content and the activity of fructosyltransferases during the growth of wheat seeds (Triticum aestivum L. Thell, cv Caldwell). The total fructan content of the seeds decreased significantly during seed growth. The trisaccharide and tetrasaccharide content increased from 6 to 28 days post anthesis (DPA) and then declined, but these changes are not statistically significant. The content or concentration of longer chain polymers did decline significantly (64.55-6.52 milligrams per gram dry weight). Free fructose also decreased significantly during seed growth indicating that the fructose liberated from the decrease in fructan content was utilized by the seed. Sucrose increased significantly from 6 to 12 DPA, then declined significantly from 12 to 28 DPA. Sucrose:sucrose fructosyltransferase activity was greatest from 6 to 12 DPA (averaging 0.16 micromole of fructose transferred per seed per hour), then declined rapidly (0.04 micromole of fructose transferred per seed per hour). The estimated activity of fructan:fructan fructosyltransferase followed a similar pattern. The increase in sucrose concentration and high enzyme activity suggests that fructans were synthesized during the lag phase of seed growth.

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PubMed1986

Sucrose synthase activity in developing wheat endosperms differing in maximum weight.

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the "rate limiting" enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.

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PubMed1979

Partitioning of C-photosynthate, and long distance translocation of amino acids in preflowering and flowering, nodulated and nonnodulated soybeans.

The influence of stage of development (preflowering versus flowering) in nodulated and nonnodulated soybeans (Glycine max [L.] Merr. cv. Wells) on partitioning of (14)C into assimilates following exposure of a soybean leaf to (14)CO(2) by both steady-state and pulse-labeling techniques was studied. Blades on the second fully expanded leaf from the stem apex were exposed to (14)CO(2). Radioactive assimilates were extracted from source leaf blades, petioles, and stems (both the path up and path down from source leaf), were separated into neutral (sugars), basic (amino acids), and acidic (organic acids, sugar phosphates) fractions by ion exchange chromatography. The basic fraction was further resolved using thin layer chromatography and the percentage of radioactivity recovered in each amino acid was determined.The distribution of radioactivity in the neutral, basic, and acidic fractions of the source leaf blades was significantly different from that of the transport path (petiole and stems). About 70% of the radioactivity in source leaf blades was recovered in the neutral fraction, whereas about 90% of the recovered radioactivity in the path was in the neutral fraction. (14)C-Aminoacids constituted 8 to 17% and 2 to 7% of the recovered radioactivity in source leaves and paths, respectively. Recovered (14)C in organic acids ranged from 13 to 20% and 2 to 7% in source leaves and paths, respectively. Partitioning of (14)C-assimilates among the neutral, basic, and acidic fractions was not affected by the presence of nodules or flowers. However, when steady-state labeling was compared to pulse labeling, a significantly lower percentage of (14)C was recovered in the neutral fraction with a concomitant increase in the basic fraction. Asparagine-arginine, serine, glutamate, gamma-aminobutyrate-alanine, and aspartate accounted for 69 to 85% of the recovered radioactivity in the basic fraction from the various treatments. [(14)C]Serine was significantly higher in pulse-labeling experiments, whereas glutamtate and proline were higher with steady-state labeling. [(14)C]Serine was significantly higher in nonnodulated plants than in nodulated plants, whereas gamma-aminobutyrate-alanine was significantly higher in preflowering plants as compared to flowering plants.

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PubMed1978

Source pool kinetics for C-photosynthate translocation in morning glory and soybean.

The kinetic behavior of translocation profiles indicates that their shape is determined largely by the rate at which tracer enters the sieve tubes in the source leaf. Confirmation of this relationship was sought by investigating the kinetics of (14)C in the immediate source pool for translocated sucrose in soybean (Glycine max L., cv. Bragg) and morning glory (Ipomea nil Roth, cv. Scarlet O'Hara) leaves. Quantitative microautoradiography was used to follow the water-soluble (14)C contents of the companion cells in minor veins after pulse-labeling with (14)CO(2). In both morning glory and soybean, the observed kinetics in the companion cells matched reasonably well those expected from the shape of the translocation profiles.Marked compartmentation of sucrose was evident in soybean leaves in that the specific radioactivity of total leaf sucrose was greatest immediately after labeling and quickly declined, whereas labeling in the companion cells was low at first and did not reach a maximum for about 35 minutes. In morning glory leaves, the kinetics of sucrose specific radioactivity and of companion cell-labeling more closely paralleled one another.

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PubMed1977

Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean.

To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. ;Wells'), (14)C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with (14)C-sucrose and (14)CO(2) assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.

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PubMed1977

Long Distance Translocation of Sucrose, Serine, Leucine, Lysine, and Carbon Dioxide Assimilates: II. Oats.

To establish whether several amino acids were equally able to enter the phloem of oat (Avena sativa L.) plants and be transported, several (14)C-labeled amino acids were applied individually to an abraded spot on a fully expanded source leaf. The base of an immature sink leaf was monitored with a GM tube for time and rate of arrival of radioactivity. Transport of (14)C-sucrose and (14)CO(2) assimilates was measured for a comparison. The applied l-serine, l-lysine, and l-leucine, as well as sucrose, entered the phloem and were transported to the sink leaf at rates between 1.16 and 1.83 cm/min. Transport velocity for CO(2) assimilates was 1.57 cm/min. A heat girdle near the top of the source leaf sheath blocked most transport, which indicated that transport was primarily through the phloem. Mass transfer rates for amino acids were only 3% as great as that for sucrose, suggesting different mechanisms of entry for sucrose than for amino acids into the phloem. The higher percentage of CO(2) assimilates mobilized to the sink leaf was attributed to the greater surface area of minor veins accessible to loading, as compared to those compounds supplied via an abraded spot. Serine was extensively metabolized in the source leaf, and radioactive products in the sink leaf mirrored those in the source leaf. Most radioactivity of lysine and leucine remained within these compounds in the source, path, and sink tissues. We concluded that there was no barrier to entry of amino acids into the phloem and transport therein. Data do not suggest a specific mechanism for entry of amino acids into the phloem.

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PubMed1977

Estimation of Osmotic Gradients in Soybean Sieve Tubes by Quantitative Autoradiography: Qualified Support for the MUnch Hypothesis.

An attempt was made to evaluate Münch's hypothesis of osmotically generated pressure flow in soybean (Glycine max L.) sieve tubes from velocity measurements and calculations of pressure potentials and sieve tube resistances. Pressure potential was estimated from values for water potentials and osmotic potential. Leaf water potential measurements were made by isopiestic thermocouple psychrometry, while the water potential of the nutrient solution was made with a vapor pressure osmometer. Osmotic potential was measured by first bringing the sucrose pools in the entire plant to the same specific radioactivity by steady-state-labeling of the shoot with constant specific radioactivity (14)CO(2) for 5 to 8 hours. Sucrose concentrations in sieve tubes were calculated from the disintegration rate per unit volume in sieve elements as measured by absolute quantitative microautoradiography of freeze-substituted, Eponembedded source (leaf) and sink (root) tissues.Conductivity of the sieve tubes was calculated from measurements of their dimensions in the petiole, stem, and root. The total pressure drop required for pressure flow at the observed velocities was calculated from the conductivity, velocity, and path length.In all experiments, the calculated sucrose concentration in source sieve tubes was greater than that in sink sieve tubes, with an average ratio (source to sink) of 1.79:1. However, the absolute sucrose concentrations (average values of 46.4 mg cm(-3) in the source and 23.9 mg cm(-3) in the sink) would have been insufficient to maintain positive turgor in the sieve elements, and the expected pressure differences would not have accounted for movement at the observed velocities. However, the low values for sucrose concentrations almost certainly were due to loss of sucrose during tissue preparation but, for technical reasons, such loss could not be accurately quantified.Assuming a sucrose concentration sufficient to maintain zero turgor in the root sieve tubes, a xylem water potential gradient (psi(w) [sink] - psi(w) [source]) of 2 bars between source and sink, and the measured ratio of sucrose concentrations in source and sink (1.79:1), the average turgor gradient between source and sink (psi(p) [sink] - psi(p) [source]) would have been about -1.6 to -3.5 bars, which compares favorably with the -1.07 to -2.41 bars average gradient that would have been required to drive translocation at the observed velocities.

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PubMed1975

The efficiency of 14C detection in autoradiographs of semithin plastic sections.

Sections of uniformly labeled 14C-methylmethacrylate were used to determine the efficiency of 14C detection by Ilford L4 emulsion layers, 0.22 mu in thickness, in light microscope autoradiographs. When efficiency was expressed as a function of radiation dose (i.e., the number of decays/mu2) there was a strong dependency on radiation dose up to about 6 decays/mu2, after which detection efficiency was fairly constant. For most emulsion batches, the detection efficiency was about 5%, but it ranged from 8 to 3%.

Autoradiography↗

PubMed1972

The Retention of Water-soluble Compounds during Freeze-Substitution and Microautoradiography.

Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated (14)C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from (14)C-sucrose-infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their (14)C-sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded (14)C-sucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining.

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