Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

Seasonal Dynamic of Nonstructural Saccharides in a Rhizomatous Grass Calamagrostis Epigeios

Seasonal dynamic of total nonstructural saccharides (TNS) and individual saccharides (starch, sucrose, glucose, fructose, fructans) was followed in rhizomes and stem bases of Calamagrostis epigeios (L.) Roth at two types of meadows communities in the South Moravia (Czech Republic): cnidion and molinion alliances, which differ in their water regime. The TNS were formed mainly by fructans and starch, while glucose, sucrose and fructose were low. The amount of TNS in rhizomes and stem bases of plants from wet cnidion site was higher than in plants from drier molinion site. The seasonal trends of all saccharides were similar in the both sites. During growing season (June to October) the main storage sugar was fructan (18 – 21 % of dry biomass). At the beginning of September the content of fructan decreased to 10 – 12 % and simultaneously the content of sucrose increased from 1 to 3 %. This may increase frost resistance. The content of TNS in the stem bases was lower than in the rhizomes. During winter time the stem bases contained 2 to 2.5 % of sucrose. Plant height and aboveground biomass were also higher in molinion site.     

Analysis of nonstructural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection

A comprehensive analysis of nonstructural carbohydrates in storage organs (bulbs and corms) of 30 ornamental geophytes was conducted by employing a variety of extraction techniques followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Among species, starch, fructan, glucomannan and soluble sugars accounted for 50-80% of storage organ dry weight (DW). Starch ranged from 24 to 760 mg g(-1) DW, fructan (commonly occurring with starch) from 25 to 500 mg g(-1) DW, and glucomannan from 15 to 145 mg g(-1) DW. An acid hydrolysis protocol for concurrent determination of fructan and glucomannan was developed. The average degree of polymerization (DP) of ethanol and water-soluble fructan and the man : glu ratio of glucomannan also varied between species. The 80% ethanol fraction contained soluble sugars and short-chain fructans (< 25 DP), whereas water extracts contained soluble sugars, fructans (both short- and long-chain, 相似文献   

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 nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare)

Seedlings of barley ( Hordeum vulgare L. cv. Agneta) were grown hydroponically under continuous light, constant temperature and relative humidity. During the first two weeks, the relative growth rate (RGR) was kept at 25% by limiting only the supply of nitrogen. The cultures were then transferred to nitrogen-free media and the amounts of fructan, starch, sucrose, glucose and fructose in sink and source leaves were measured at 0, 12, 24, 48, 72, 120 and 156 h. The activities of two key enzymes in fructan metabolism, sucrose:sucrose fructosyltransferase (SST), fructan exohydrolase (FEH), as well as acid invertase were also measured in the two types of leaves.
The fructan and starch levels in both sink and source leaves increased during nitrogen deficiency. The highest increase in starch was 200% of the control while for fmctans a 700% increase was recorded. The activity of SST increased parallel to fructan accumulation in sink leaves. However the FEH activity was constant and not affected by nitrogen deficiency. The invertase activity both in sink and source leaves was reduced by nitrogen deficiency. More fructans as well as sucrose and fructose accumulated in source leaves compared to sink leaves both before and after nitrogen starvation. The results show that fructan is the major carbohydrate reserve accumulating under nitrogen deficiency both in sink and source leaves in barley plants. The induction of fructan accumulation in sink leaves caused by nitrogen deficiency is intimately connected with the regulation of SST     

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.     

Changes in non-structural carbohydrate composition during bulbing in sweet and high-solid onions in field experiments

The composition of non-structural carbohydrate (NSC) content of predominantly long-day onion germplasm has been assessed over several years and in a bi-location trial. It was observed that genetic rather than environmental factors determined the NSC composition of onion bulbs. Glucose was the NSC component which was most closely correlated with genotype. Fructose was the only NSC component that was significantly affected by environment. Sucrose and 1-kestose (DP3 fructans) were not correlated to a large extent to the other NSC components, indicating their transient role in the fructan metabolism. Strong negative correlations were observed between reducing sugars (i.e. fructose and glucose) and dry matter content (DM). Furthermore, it was shown that accessions differed significantly in their fructan accumulation pattern; high DM accessions showed accumulation of fructans over the whole bulbing period, whereas, low DM accessions quickly reached a plateau. Implications for the breeding of high quality onions are discussed.     

Diurnal carbohydrate metabolism of barley primary leaves

The carbohydrate content of barley (Hordeum vulgare L.) leaves was measured over a 24-hour cycle. Nonstructural carbohydrate accumulation was linear after the 1st hour of light, whereas utilization in the dark was fast initially and slowed as stored reserves were depleted. Sucrose was the most abundant storage form of carbohydrate in the primary leaf. Lesser amounts of starch, fructans, and hexoses were also present. Leaf reserves were almost completely remobilized by the end of the dark period. There was a lag in starch degradation following a light to dark transition. Lower rates of starch accumulation were observed at the beginning and at the end of the day. Fructan synthesis occurred primarily towards the end of the light period as rates of sucrose and starch synthesis decreased. The above results suggested that carbohydrate metabolism in primary barley leaves was controlled by light and by endogenous factors such as foliar sucrose levels. Measurements of specific [¹⁴C]sucrose activity in steady state labeled 7-day-old barley primary leaves suggested the presence of at least two kinetically separate pools. Sucrose levels were higher and apparent turnover rates were lower in barley leaves in comparison to previous studies with other species.     

Carbohydrate Status of Tulip Bulbs during Cold-Induced Flower Stalk Elongation and Flowering

The effect of a cold treatment on the carbohydrate status of the scales and flower stalk of Tulipa gesneriana L. cv Apeldoorn bulbs during growth after planting was studied and compared with bulbs not given cold treatment. Bulbs were stored dry for 12 weeks at 5[deg]C (precooled) or 17[deg]C (noncooled). Only the 5[deg]C treatment led to rapid flower stalk elongation and flowering following planting at higher temperatures. Precooling enhanced mobilization of starch, fructans, and sucrose in the scales. The cold-stimulated starch breakdown was initially accompanied by increased [alpha]-amylase activity per scale. In noncooled bulbs, [alpha]-amylase activity slightly decreased or remained more or less constant. Cold-induced flower stalk elongation was partially accompanied by a decrease in the sucrose content and an increase in the glucose content and invertase activity per g dry weight. The starch content in internodes initially decreased and subsequently increased; [alpha]-amylase activity per g dry weight of the lowermost internode showed a peak pattern during starch breakdown and increased thereafter. The internodes of noncooled bulbs, on the contrary, accumulated sucrose. Their glucose content and invertase activity per g dry weight remained low. Starch breakdown was not found and [alpha]-amylase activity per g dry weight of the lowermost internode remained at a low level. Precooling of tulip bulbs thus favors reserve mobilization in the scales and flower stalk and glucose accumulation in the elongating internodes.     

Changes of enzyme activities associated with the mobilization of carbohydrate reserves (fructans) from the stem of wheat during kernel filling

Wheat plants were grown at a day/night temperature of 18/13°C under glasshouse conditions. Twenty-two d after anthesis, one set of plants was shaded to 50% of the normal photon fluence rate, another was 'degrained' by selective spikelet removal which left only the grains in the five central spikelets; a further set was left as control. Individual plants were harvested at days 22, 30 or 42 after anthesis. Extracts from the peduncle and the penultimate internode were prepared to determine the activities of sucrose phosphate synthase, sucrose synthase, fructan exohydrolase and acid invertase, and to assess the concentration of hexose sugars, sucrose and fructans. Measurements were also made of ear and individual grain weights, and stem f. wt and d. wt. There was a decline in the amount of fructans with time, more pronounced in 'shaded' (source-limited) than in control plants. By contrast, in 'degrained' (sink-limited) plants, the amount of fructans in the stem initially rose, then decreased, with a concomitant increase in the amount of fructose. The shifts in sugar content of the wheat culm reflected both the sink demand of the ear and source activity. The activity of fructan exohydrolase correlated with the carbohydrate changes. Under limited photosynthate assimilation, the mobilization of fructans from the internodes towards the ear was related to an increase in this enzyme, whereas the other enzymes played a less direct role in the mobilization of fructan reserves from the wheat stem.     

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.     

Fructose-containing carbohydrates in the tuberous root of Gomphrena macrocephala St.-Hil. (Amaranthaceae) at different phenological phases

Gomphrena macrocephala St.-Hil. (Amaranthaceae) is a perennial herb that grows spontaneously in the cerrado and is characterized by well-defined phenological phases throughout the year. Soluble carbohydrates are the main reserve compounds of the tuberous root and constitute approximately 50% of the dry weight. These sugars were partially characterized as fructans forming a single homologous series, different from inulin, the most common fructan of dicotyledons. The mean molecular weight of polysaccharides was high and reached 37 kDa in the dormant phase. Fructan spherocrystals were detected in the tuberous root after treatment with ethanol, being associated with the parenchyma of secondary xylem. The content, composition and mean molecular weight of fructans were related to phenology. In late dormancy, there was a marked increase in monosaccharides, particularly fructose, and a concomitant decline of polysaccharides, probably as a result of fructan breakdown. During sprouting and in the vegetative phase, the contents of oligosaccharides and low molecular weight polysaccharides increased. A gradual rise in the molecular weight of polysaccharides occurred during the reproductive phase and at early dormancy, concurrently with decreasing levels of oligosaccharides. The capacity of G. macrocephala to accumulate readily accessible sugars, such as fructans, instead of starch, in response to environmental changes, may be of considerable advantage, since the cerrado is often subjected to seasonal drought and burnings.     

Exogenous nitric oxide effect on fructan accumulation and FBEs expression in chilling-sensitive and chilling-resistant wheat

This study was to investigate the effect of exogenous nitric oxide (NO) on fructan accumulation and fructan biosynthesic enzymes (FBEs) expression in seedlings leaves of two wheat (Triticum aestivum L.) cultivars, winter wheat (Zhoumai18, ZM) and spring wheat (Yanzhan4110, YZ), under 4 °C. The seedlings of two wheat cultivars were subjected to different concentrations of sodium nitroprussiate (SNP) for 0, 24, 48, and 96 h. Relative water content (RWC) was increased by exogenous NO in YZ, but decreased in ZM. Except for glucose, fructose and fructans of degree of polymerization (DP) 3 in YZ, other soluble carbohydrates contents in the two wheat cultivars all increased to different degrees. The activities of FS (including sucrose: sucrose 1-fructosyltransferase (1-SST, EC: 2.4.1.99) and sucrose: fructan 6-fructosyltransferase (6-SFT, EC: 2.4.1.10)) were significantly higher than fructan: fructan 1-fructosyltransferase (1-FFT, EC: 2.4.1.100) in the seedlings of two wheat cultivars. The same phenomenon occurred to FBEs expression. In addition, sucrose content decreased while fructans content increased under low temperature, which was in accordance with the improved 1-FFT activity in ZM. Moreover, fructans content increased to a high level under high concentration of NO in ZM while kept at a constant low level in YZ. The expression levels of FBEs were universally higher in ZM than in YZ, which identified with the high frost resistance of the winter cultivar. It is concluded that exogenous NO treatment on wheat may be a good option to reduce chilling injury by regulating fructan accumulation in leaves. This is the first report owing that exogenous NO alleviated the negative effects of chilling stress by accumulating fructans in wheat.     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance

The effect of cold and abscisic acid (ABA) treatment on soluble carbohydrate content was compared in callus cultures of wheat genotypes differing in frost tolerance. The effect of 5A chromosome substituted from the frost tolerant to the sensitive on cold-induced carbohydrate accumulation was also determined. Following cold hardening, the increase in sucrose and fructan level in calli of tolerant varieties was significantly higher than those of the sensitive ones. In 5A substitution line higher sucrose and fructan content was detected than in recipient . Tendentiously, cold stress caused higher degree of changes in carbohydrate content than the exogenously applied ABA did. Comparing the accumulation pattern of the components of WSC measured in vitro to the previously published in vivo results it can be concluded that in the case of sucrose and fructans it was similar, while for the reducing sugars it was different. The regulatory role of chromosome 5A either in the development of freezing tolerance or carbohydrate accumulation was confirmed in dedifferentiated calli, as well.     

Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.     

Sucrose and fructan metabolism in wheat roots at chilling temperatures

Sucrose and fructan metabolism were studied in wheat ( Triticuin aotiirum L. cv. Tribal 800) roots during a period at chilling temperature. Enzyme activities related to fructan and sucrose metabolism were measured. Sucrose-sucrose fructosyl transfer-ase (EC 2.4.1.99) activity increased more than 25-fold when plants were cooled to 4°C. Sucrose synthase (EC 2.4.1.13) and sucrose-phosphate synthase (EC 2.4.1.14) activities also increased, but low temperatures had no significant effect on invertaso (EC 3.2.1.26) or on fructan hydrolase (EC 3.2.1.26) activities. The accumulation pattern of fructan in roots was different to that in leaves. In roots chilling stimulated the synthesis of fructans of high degree of polymerization.     

Reuteran and levan as carbohydrate sinks in transgenic sugarcane

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03?mg/g FW), sucrose content (ca 60?mg/g FW) was not affected, while starch content (<0.4?mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01?mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.