Pathway of fructosan synthesis in leaf bases of Dactylis glomerata

Chemical analysis of leaf base tissue of Dactylis glomerate failed to detect any low MW oligosaccharide intermediates during fructosan synthesis. Extracts of tissue harvested at various times after the incorporation of ¹⁴CO₂ showed a decline in radioactivity in sucrose and an equivalent rise in high MW fructosan with no significant accumulation of radioactivity in oligosaccharides. No evidence was obtained for the existence of nucleotide fructose in the tissue, indicating that fructosan synthesis occurs by direct transfer of fructosyl residues from sucrose to the polymer.     

Wax synthesis in Brassica oleracea as modified by trichloroacetic acid and glossy mutations

Two different mutations in Brassica oleracea, gl₅ and gl₄ have been re-investigated using acetate-1-¹⁴C labelling in an attempt to define more closely the nature of the genetic blocks to wax synthesis. It has been found that gl₅ is a mutation which blocks elongation in the Step C₂₈–C₃₀. The mutation gl₄ exhibits no elongation block and could be blocked in the decarboxylation Step C₃₀–C₂₉. 0·1 mM TCA supplied in the culture solution of cauliflower seedlings affected the leaf surface by producing a glossy appearance similar to that induced by gl₃ and gl₄. At this concentration growth was not inhibited and the appearance of the plants was normal except for the surface wax. The amount of surface wax produced was about 40% of that in untreated seedlings on a leaf area basis. Slight, but significant changes in wax composition were noted, mainly involving a reduction in C₃₀ acids and aldehydes, a slight reduction (33–29%) in alkane content, and a marked difference in chain length composition of the alkanes with C₂₇ increased relative to C₂₉. Over a range of concentrations from 0·1–1 mM, TCA inhibited incorporation of label from acetate-1-¹⁴C into C₃₀ acids and aldehydes more than into C₂₈ at concentrations 0·4–0·8 mM while label tended to accumulate in C₂₄ and C₂₆ acids; thus elongation C₂₈–C₃₀ was especially sensitive to TCA. TCA also inhibited incorporation into primary alcohols and esters almost as much as into C₂₉ compounds. In spite of relatively specific effects on incorporation of label into longer chain lengths, the resulting block to C₃₀ synthesis is not sufficient to make much difference to the overall rate of C₂₉ synthesis. Both results of analysis of wax from whole plants and experiments with tissue slices in vitro indicated that the effect of TCA in reducing the glaucousness of the leaf surface is a combination of overall reduction of wax synthesis together with slight but significant changes in wax composition.     

Effects of the Fungal Endophyte Acremonium coenophialum on Nitrogen Accumulation and Metabolism in Tall Fescue

Infection by the fungal endophyte Acremonium coenophialum affected the accumulation of inorganic and organic N in leaf blades and leaf sheaths of KY 31 tall fescue (Festuca arundinacea Schreb.) grown under greenhouse conditions. Total soluble amino acid concentrations were increased in either the blade or sheath of the leaf from infected plants. A number of amino acids were significantly increased in the sheath, but only asparagine increased in the blade. Infection resulted in higher sheath NH₄⁺ concentrations, whereas NO₃⁻ concentrations decreased in both leaf parts. The effects on amino acid, NO₃⁻, and NH₄⁺ concentrations were dependent upon the level of N fertilization and were usually apparent only at the high rate (10 millimolar) of application. Administration of ¹⁴CO₂ to the leaf blades increased the accumulation of ¹⁴C in their amino acid fraction but not in the sheaths of infected plants. This may indicate that infection increased amino acid synthesis in the blade but that translocation to the sheath, which is the site of fungal colonization, was not affected. Glutamine synthetase activity was greater in leaf blades of infected plants at high and low N rates of fertilization, but nitrate reductase activity was not affected in either part of the leaf. Increased activities of glutamine synthetase together with the other observed changes in N accumulation and metabolism in endophyte-infected tall fescue suggest that NH₄⁺ reassimilation could also be affected in the leaf blade.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves

Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline—remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.

When [methyl-¹⁴C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more ¹⁴C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [¹⁴C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more ¹⁴C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [¹⁴C]formate for about 18 hours in darkness, betaine was always the principal ¹⁴C-labeled soluble metabolite. This ¹⁴C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of ¹⁴C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [¹⁴C]formate.

These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.

     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.     

Role of nitrogen metabolism in the development of salt tolerance in barley plants

The 7- to 8-day-old barley (Hordeum vulgare L.) seedlings grown in KNO3 solutions (1-40 mM) were characterized by the substrate activation of nitrate reductase (NR) in the apical leaf segments (1–2 cm in length), as well as by stimulated growth, broadened leaf blades, and by vigorously developed system of shortened roots. When the seedlings were grown in the presence of 20 mM KNO₃, the ability of leaf segments to generate superoxide anion radical remained at the level typical of control plants grown in water. The content of 5-aminolevulinic acid (ALA) in plants grown in the presence of 20 mM KNO₃ was 2.2–2.4 times higher than in control plants. The plants grown in the presence of nitrate had an elevated content of chlorophylls a and b, heme, and protein (by 42%). At the same time, the proline content was almost twofold lower than in control plants, which was due to substantial reduction (by 40%) in activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS). It is concluded that the substrate activation of NR by KNO₃ under normal growth conditions results in predominant utilization of glutamic acid (the primary product of inorganic nitrogen assimilation) for biosynthesis of tetrapyrroles and protein amino acids at the expense of inhibition of proline synthesis. When barley seedlings were grown in 150 mM NaCl solution, the plant growth and the root system development were suppressed to the levels of 63 ± 6% and 61 ± 11% of the control values, respectively. In the apical leaf tissues of plants adapted to NaCl, there was a slight decrease in the total NR activity (by 10%), a significant reduction in protein content (by 32%), and a parallel increase in the content of ALA (by a factor of 4.3), chlorophylls, heme, carotenoids, proline (2.2-fold) and P5CS (1.6-fold) with respect to the control values. It is proposed that the accumulation of ALA and proline under salinity-induced suppression of nitrogen assimilation results from the predominant allocation of glutamate for biosyntheses of ALA and proline at the expense of inhibition of growth-related processes requiring intense protein synthesis. The substrate activation of NR by KNO₃ under salinity conditions was associated with prevailing allocation of the assimilated nitrogen for synthesis of proline and protein amino acids, which reinforced plant cell protection against salinity and stimulated plant growth.     

Fructosan metabolism in Cichorium intybus roots

Incorporation of [¹⁴C]sucrose into difructosyl glucose (F₂G), trifructosyl glucose (F₃G) and tetrafructosyl glucose (F₄G) in the presence of various nucleoside triphosphates revealed that formation of F₄G and F₃G is retarded in the presence of ATP, and formation of F₃G and F₂G is significantly enhanced in the presence of CTP, whereas UTP has no effect on the synthesis of these oligosaccharides. Different fructosyl transferases seem to be responsible for the different fructosylation steps and self transfer seems to be the major pathway for fructosan synthesis. Utilization of added glucose, which is formed by sucrose sucrose fructosyl transferase action in vivo, is completely inhibited in acetate buffer whereas in phosphate, citrate and citrate-phosphate buffers glucose is actively utilized. In the presence of fluoride ions both glucose utilization and its conversion to CO₂ is inhibited by ca 50%. CO₂ production from [¹⁴C]glucose is completely inhibited in acetate ions. No evidence for the incorporation of ¹⁴C from [¹⁴C]glucose into [¹⁴C]sucrose is observed. The ratio of bound fructose to bound glucose is the same in the entire length of the root indicating that there is no preferential zone for fructosan synthesis.     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA₁ under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA₁ uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.     

Root-zone CO2 and root-zone temperature effects on photosynthesis and nitrogen metabolism of aeroponically grown lettuce (Lactuca sativa L.) in the tropics

Effects of elevated root-zone (RZ) CO₂ concentration (RZ [CO₂]) and RZ temperature (RZT) on photosynthesis, productivity, nitrate (NO₃ ^?), total reduced nitrogen (TRN), total leaf soluble and Rubisco proteins were studied in aeroponically grown lettuce plants in a tropical greenhouse. Three weeks after transplanting, four different RZ [CO₂] concentrations (ambient, 360 ppm, and elevated concentrations of 2,000; 10,000; and 50,000 ppm) were imposed on plants at 20°C-RZT or ambient(A)-RZT (24–38°C). Elevated RZ [CO₂] resulted in significantly higher light-saturated net photosynthetic rate, but lower light-saturated stomatal conductance. Higher elevated RZ [CO₂] also protected plants from both chronic and dynamic photoinhibition (measured by chlorophyll fluorescence Fv/Fm ratio) and reduced leaf water loss. Under each RZ [CO₂], all these variables were significantly higher in 20°C-RZT plants than in A-RZT plants. All plants accumulated more biomass at elevated RZ [CO₂] than at ambient RZ [CO₂]. Greater increases of biomass in roots than in shoots were manifested by lower shoot/root ratios at elevated RZ [CO₂]. Although the total biomass was higher at 20°C-RZT, the increase in biomass under elevated RZ [CO₂] was greater at A-RZT. Shoot NO₃ ^? and TRN concentrations, total leaf soluble and Rubisco protein concentrations were higher in all elevated RZ [CO₂] plants than in plants under ambient RZ [CO₂] at both RZTs. Under each RZ [CO₂], total leaf soluble and Rubisco protein concentrations were significantly higher at 20°C-RZT than at A-RZT. Our results demonstrated that increased P _Nmax and productivity under elevated [CO₂] was partially due to the alleviation of midday water loss, both dynamic and chronic photoinhibition as well as higher turnover of Calvin cycle with higher Rubisco proteins.     

Sea urchin egg fertilization studied with a fluorescent probe (ANS)

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10^?14 g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Translocation of sucrose by squash plants

Sucrose-¹⁴C was fed to the leaf blades of squash plants (Cucurbita pepo L. var. melopepo torticallis Bailey) for 30 or 60 minutes. Petioles of treated plants were cut into sections and extracted. The majority of the ¹⁴C within the petiole was in sucrose rather than stachyose, the sugar that is transported by the squash plants when ¹⁴CO₂ is supplied. This indicates that the phloem loading system of squash plants is not the system that specifies which sugar is transported.     

Studies on fructosyl transferase from Agave americana

The possible role of fructosyl transferase in the biosynthesis of fructosans in Agave americana was investigated. This enzyme was extracted from A. americana stem and purified 17.5-fold by salt fractionation and DEAE-cellulose chromatography. The optimum conditions for the enzyme were pH 6. 1, temperature 37°, substrate concentration 20% and K_m 3.6 × 10^?1 M; Ag⁺, Pb ²⁺, Hg²⁺, Al³⁺, Sn²⁺, CN^? acted as inhibitors and Ca²⁺, Mg²⁺, Co²⁺ and Li⁺ actemd as activators. Only sugars of the type F ～ R (R-aidose), e.g. sucrose and raffinose acted as substrates for the enzyme. The donor acceptor specificity of the enzyme was studied extensively. Sugars sucrose. None of the intermediates of fructosan biosynthesis from sucrpse acted as fructose donors. The possible acceptors from sucrose and raffinose. The enzyme was capable of building up oligosaccharides up to FIOG from sucrose. None of the intermediates of fructosan biosynthesis from sucrose acted as fructose donors. The possible mechanism of fructosan biosynthesis from sucrose is discussed.     

Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb

Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with ¹⁴CO₂ was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

Glycine as a precursor of bacterial purines

The synthesis of purines in Aerobacter aerogenes was studied by the use of labeled glycine [CH₂(N¹⁵H₂)C¹³OOH]. Adenine and guanine were isolated from the lyophilized cell material. The isotopes were found in the bases. Evidence for the incorporation of the whole glycine molecule is presented.     

Photosynthesis and respiration in a fast growing strain of Gigartina exasperata (Harvey and Bailey)

Photosynthesis and respiration rates of blades from a selected, fast growing strain of the marine red alga. Gigartina exasperata Harvey and Bailey, a carrageenan producer, were measured with an oxygen electrode and compared with rates similarly obtained from wild material of the same species. The measurements, expressed as μl O₂ · mg chl a^?1, min^?1. were made over a light intensity range from 5 to 800 μE · m^?2 · sec^?1 and a temperature range of 6 to 16°C. The photosynthesis light intensity data are best described by hyperbolic functions.     

Carbon balance of Yucca elata Engelm. during a hot and cool period in situ

Respiratory activity of intact, attached roots was measured under field and controlled conditions. Root respiration of Yucca elata Engelm. was highly temperature dependent: Q₁₀ values decreased from 2.1 (12–22° C) to 1.7 (26–36° C) as temperatures increased. Respiration ceased after 5 h at 42° C. In the field, in August, when net leaf photosynthesis was severely depressed, the diel fluctuation in the respiration rate of suberized and partially suberized roots was predominantly a function of temperature. A photoperiod-associated rise in respiration rates apart from temperature response occurred in February for nonsuberized, partially suberized, and suberized roots when active net photosynthesis occurred throughout the photoperiod. In whole-plant root systems, respiratory CO₂ was 3.2 and 4.3 mg CO₂·g DW^-1·d^-1 in August and February, respectively, when adjusted for the proportion of suberized and nonsuberized lateral roots. On a whole-plant basis, 0.89 mg C·g DW^-1·d^-1 was gained during February and 0.46 mg C·g DW^-1·d^-1 was lost in August. The belowground: aboveground ratio of whole-plants in situ was 0.42 on a shallow soil where vertical root growth was limited to a soil depth of 68 cm and ranged from 1.29 to 5.94 \(\left( {\bar x = 3.31} \right)\) in deep sands. No leaf dark fixation of CO₂ was observed in field plants during August and February, nor in well-watered plants or plants subjected to drought in laboratory studies. Although small diel fluctuations in leaf acidity occurred in both field and greenhouse-grown plants, results of this study suggest that Y. elata is a C₃ plant.