Andrew J. Cairns

Glycosidase-inhibiting pyrrolidine alkaloids from Hyacinthoides non-scripta

Abstract The glycosidase-inhibiting pyrrolidine alkaloids (2 R ,3 R ,4 R ,5 R )-2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), 2,5-dideoxy-2,5-imino- dl - glycero - d - manno -heptitol (homoDMDP), homoDMDP-7- O -apioside and 1,4-dideoxy-1,4-imino- d -arabinitol have been identified in the leaves of bluebells ( Hyacinthoides non-scripta ). HomoDMDP and homoDMDP-7- O -apioside are new natural products. Glycosidase inhibition by the aglycones is compared and could explain the symptoms of poisoning of livestock by bluebells.

Direct effects of low temperature upon components of fructan metabolism in leaves of Lolium temulentum L.

Summary Specific effects of temperature upon fructan metabolism were measured in intact leaves of Lolium temulentum L. held at 5°C by comparing them with leaves where fructan accumulation at 20 °C had been induced by excision. Changes in fructose 2,6-bisphosphate contents differed markedly, with leaves at 5 °C showing a sustained decrease and excised leaves a marked increase, even though both treatments caused increased rates of sucrose accumulation in the tissue. Activity of cytoplasmic fructose-1,6-bisphosphatase, which is inhibited by fructose-2,6-bisphosphate, remained constant for 8 h following excision but increased by 64 % in chilled leaves. Measurements of incorporation of 14 CO 2 into oligosaccharides showed that chilled and excised leavesdiffered in the patterns of accumulation of radioactivity in trisaccharide isomers and the relative abundance of labelled tetra- and pentasaccharides, but not in the progressive movement of radioactivity from sucrose to high molecular weight fructan. Low incubation temperatures also affected the relative proportions of the different isomeric trisaccharides produced in vitro by crude SST and purified invertase preparations supplied with sucrose, mainly by increased accumulation of kestose. These results are discussed in relation to the effects of different environmental conditions during the induction of fructan synthesis in leaves of temperate gramineae.

Comparison of growth and carbohydrate accumulation in seedlings of two varieties of Lolium perenne

Turner, L. B., Humphreys, M. O., Cairns, A. J., Pollock, C. J. (2001). Comparison of growth and carbohydrate accumulation in seedlings of two varieties of Lolium perenne. Journal of Plant Physiology, 158, (7), 891-897. Sponsorship: BBSRC RASP Initiative

Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: Applicability to the measurement of fructosyl transferase activity in higher plants

A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Abstract. Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Starch metabolism in the fructan-grasses: Patterns of starch accumulation in excised leaves of Lolium temulentum L.

Summary Excised and illuminated leaves of temperate gramineae have previously provided a powerful system for the analysis of the regulation of partitioning of photosynthate between sucrose and fructan. This paper complements previous data by describing the concomitant accumulation of transitory starch, an assimilate which has been ignored in this system. Leaves of Lolium temulentum were depleted to near-zero reserve carbohydrate content. In this condition, the tissue contained no detectable fructan or starch and only a trace of sucrose. When subsequently excised and illuminated continuously, the accumulation of sucrose and starch commenced immediately and occurred simultaneously. Starch anabolism was constitutive and the polymer was apparently synthesised de novo . The maximal rate of starch accumulation, at 0.6 mg g −1 fresh mass, was 5-10-fold lower than that of sucrose. Fructan was also synthesised de novo and was induced after a lag of 8 h, after which rates of net sucrose and starch accumulation slowed, ceasing completely by 14-16 h. Whilst total non-structural carbohydrate concentration increased continuously, reaching 56 mg g −1 at 30 h, starch concentration was asymptotic and was limited to a maximum of only 7 mg g −1 reached at 14-16 h. Hence, net starch accumulation was not restricted by the continued production of photosynthate. The illumination period spanned 1.25 diurnal cycles. Despite continued photoassimilation, there was no resumption of net starch synthesis at the beginning of the second cycle and hence the limitation of net starch accumulation was not imposed by an endogenous circadian rhythmicity. When assimilate partitioning into fructan was abolished by transpirational feeding of cycloheximide, sucrose concentration increased 2.7-fold to 49 mg g −1 . This abnormally high concentration did not feedback to enhance starch accumulation. Transpirational feeding of 10 mmol/L mannose inhibited sucrose accumulation by c .90 %, but this inhibition was not accompanied by an increase net starch accumulation, rather, starch accumulation was inhibited by 60 %. Mannose caused the formation of maltose in the tissue. Starch synthesis, in common with sucrose and fructan syntheses, was shown to be localised predominantly in mesophyll cells where presumably the control of partitioning between these three assimilates is exercised. The low rates, restricted tissue concentration, sucrose-insensitivity and mannose-inhibition of starch synthesis differ from many species where starch is the primary reserve. Although the available information is limited, some of these features are in common with other fructan grasses. The low capacity for starch accumulation provides a possible explanation for the evolution of fructan synthesis in these species.

Structure of fructans from excised leaves of New Zealand flax

The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g(-1) fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.

Effects of enzyme concentration on oligofructan synthesis from sucrose

Abstract At low concentrations of partially purified fructosyltransferase, only trisaccharide products were detected. At higher enzyme concentrations, larger oligofructans were detected. Depending upon enzyme concentration, the results indicated either sucrose:sucrose fructosyltransferase activity exclusively, or a more complex fructan polymerizing system. Consequences for the interpretation of fructosyltransferase data are discussed.

Production of butanol by Clostridium puniceum in batch and continuous culture

SummaryThe pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.

Carbon assimilation and partitioning into non-structural carbohydrate in contrasting varieties of Lolium perenne

Summary Rates of photosynthetic carbon assimilation and the partitioning of carbon into different sugar fractions have been compared in Aurora and Perma, two varieties of perennial ryegrass ( Lolium perenne L.). Perma had a higher photosynthetic rate than Aurora at all irradiances, and numerically a considerably higher apparent maximum quantum yield although this was not statistically significant. On a leaf area basis Perma had heavier leaves that contained more chlorophyll a , chlorophyll b and carotenoid. Carbohydrate was predominantly present in the leaves of both varieties as sucrose and fructan. Fructose, glucose and starch were undetectable or only present in trace amounts. Perma leaf blades and sheaths contained more sucrose and more small (DP3) fructan than Aurora, but less polymeric fructan. Following depletion of carbohydrate pools in low light, excised leaves were induced to accumulate fructan at high light. Perma accumulated 88 % more water-soluble carbohydrate and 90 % more starch than Aurora. However after 24 h the polymeric fructan content of Perma leaves was still lower than that of Aurora. The increase in carbohydrate content of Aurora leaves was nearly all explained by the increase in polymeric fructan, whereas Perma partitioned less into polymeric fructan and contained significantly more glucose, sucrose and DP3 fructan. Additionally, the average chain length of Perma polymeric fructan was shorter than that of Aurora.