MIR spectroscopic analysis on sugar metabolic and ethanol productive kinetics of suspension TBY-2 and rice cells pre-cultured in various media

The influence of sugars in pre-cultivation media suspended plant cells on the kinetics of the sugar uptake and the ethanol production was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells and Oryza sativa L., Japonica, cv. Nipponbare (rice) cells, respectively, in pre-culture and culture media, which had various types of glucose, fructose, sucrose or glucose–fructose mixtures. The results confirmed the kinetic differences between the TBY-2 cells and rice cells. These results suggested that the TBY-2 cells consumed sugar before growth and the rice cells consumed sugar after growth, moreover, the ethanol content increased just after cell growth was activated based on the non-dimensional cultivation time for the cell growth behavior.     

Analysis of kinetic uptake phenomena of monosaccharide and disaccharide by suspension TBY-2 cells using an FT-IR/ATR method

The influence of sugars in culture media on the kinetics of the mono- and disaccharide uptake and cell growth behavior was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells in the culture media, which contained glucose, fructose, mannose, galactose, sucrose, trehalose, maltose or lactose. Consequently, the differences of the kinetic sugar uptake and cell growth behavior among all the cultivations were confirmed. In particular, a very long lag time before the galactose uptake was observed, and the spectral-pattern of the maltose medium presented almost the same as the initial one during the cultivation. Furthermore, base on the non-dimensional cultivation time for cell growth behavior, it was suggested that the TBY-2 cells consumed sugar before cell growth and produced the ethanol just after cell growth.     

Sugar metabolic analysis of suspensions of plant cells using an FT-IR/ATR method

A simple, rapid and accurate evaluation of the sugar uptake rate of suspended plant cells from culture media was developed with the predicted sugar contents measured by mid-infrared spectroscopy using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We performed plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) in culture media, which had various combinations of glucose, fructose and sucrose concentrations at the initial stage, and measured simultaneously each sugar content in the medium by the FT-IR/ATR method. By applying a logistic function to the predicted sugar contents and cell density in the medium during cultivation, the specific sugar uptake rates by the suspended TBY-2 cells were easily and continuously obtained. Thus the kinetic sugar uptake phenomena by the TBY-2 cells were well confirmed overall using the developed method. Additionally it was found that the fraction of sucrose of the initial total sugar content might kinetically affect the sugar uptake process and cell growth. Also, the relationship between the nondimensional cell density and sucrose content could be classified into three groups on the basis of the initial fraction of sucrose.     

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

The rate of phloem loading, its selectivity, and the disposition of labeled carbon were studied following application of (14)C-labeled sugars to the free space of source leaves of sugar beet (Beta vulgaris L.). Buffered 10 mm solutions of (14)C-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 micro-densitometry 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 (14)CO(2) was studied by vacuum infiltration of the source leaf air spaces and removal of the solution by centrifugation. 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.     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Erythritol metabolism in wild-type and mutant strains of Schizophyllum commune

Erythritol uptake and metabolism were compared in wild-type mycelium and a dome morphological mutant of the wood-rotting mushroom Schizophyllum commune. Wild-type mycelium utilized glucose, certain hexitols, and pentitols including ribitol, as well as d-erythrose, erythritol, and glycerol as sole carbon sources for growth. The dome mutant utilized all of these compounds except d-erythrose and erythritol. Erythritol- or glycerol-grown wild-type mycelium incorporated erythritol into various cellular constituents, whereas glucose-grown cells lagged considerably before initiation of erythritol uptake. This acquisition was inhibited by cycloheximide. Dome mycelium showed behavior similar to wild-type in uptake of erythritol after growth on glucose or glycerol, except that erythritol was not further catabolized. Enzymes of carbohydrate metabolism were compared in cell extracts of glucose-cultured wild-type mycelium and dome. Enzymes of hexose monophosphate catabolism, nicotinamide adenine dinucleotide (NAD)-dependent sugar alcohol dehydrogenases, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-coupled erythrose reductase were demonstrated in both. The occurrence of erythrose reductase was unaffected by the nature of the growth carbon source, showed optimal activity at pH 7, and generated NAD phosphate and erythritol as products of the reaction. Glycerol-, d-erythrose-, or erythritol-grown wild-type mycelium contained an NAD-dependent erythritol dehydrogenase absent in glucose cells. Erythritol dehydrogenase activity was optimal at pH 8.8 and produced erythrulose during NAD reduction. Glycerol-growth of dome mycelium induced the erythritol uptake system, but a functional erythritol dehydrogenase could not be demonstrated. Neither wild-type nor dome mycelium produced erythritol dehydrogenase during growth on ribitol. Erythritol metabolism in wild-type cells of S. commune, therefore, involves an NADPH-dependent reduction of d-erythrose to produce erythritol, followed by induction of an NAD-coupled erythritol dehydrogenase to form erythrulose. A deficiency in erythritol dehydrogenase rather than permeability barriers explains why dome cannot employ erythritol as sole carbon source for mycelial growth.     

Carbon-use efficiency in green sinks is increased when a blend of apoplastic fructose and glucose is available for uptake

Understanding how green sink strength is regulated in planta poses a difficult problem because non-structural carbohydrate (NSC) levels can have integrated, simultaneous feedback effects on photosynthesis, sugar uptake, and respiration that depend on specific NSC moieties. Photosynthetic gametophytes of the fern Ceratopteris richardii provide a simple land plant model to assess how different NSCs imported from the apoplast of intact plants affect green sink strength. Sink strength was quantified as the amount of exogenous sugar that plants grown in low light depleted from their liquid media, and the relative contributions of carbon assimilation by photosynthesis and sugar uptake was estimated from stable isotope analysis of plant dry mass. Gametophytes absorbed fructose and glucose with equal affinity when cultured on either hexose alone, or in the presence of an equimolar blend of both sugars. Plants also depleted sucrose from the surrounding media, although a portion of this disaccharide that was hydrolysed into fructose and glucose by putative cell wall invertase activity remained in the media. The δ(13)C in plant dry masses harvested from sugar treatments were all close to -18‰, indicating that 25-39% of total plant carbon was from C3 photosynthesis (δ(13)C=-29‰) and 61-75% was from uptake of exogenous sugars (δ(13)C=-11‰). Carbon-use efficiency (i.e. carbon accumulated/carbon depleted) was significantly improved when plants had a blend of exogenous sugars available compared with plants grown in a single hexose alone. Plants avoided complete down-regulation of photosynthesis even though a large excess of exogenous carbon fluxed through their cells.     

Growth of Cephalotaxus harringtonia plant-cell cultures

Summary Cell cultures of Cephalotaxus harringtonia were examined to characterize growth kinetics. The requirement for an undefined medium supplement (coconut water) was eliminated by maintaining high cell concentrations in semicontinuous and batch growth. Sucrose fed to batch-cultured cells was completely hydrolyzed and a diauxic growth pattern was observed corresponding to first glucose and then fructose uptake. Examination of increases in cell concentrations on the basis of fresh and dry weight showed that a substantial lag period existed between the initiation of substrate uptake and increases in cell volume. Specific growth rates were highest during periods of glucose uptake, but cell yields were comparable for the two sugars. In contrast, studies with glucose or fructose as the sole carbon source indicated that cell yields were significantly lower with fructose but specific growth rates were comparable for the two sugars.Offprint requests to: P. J. Westgate     

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.     

Mutation of Clostridium thermocellum in the presence of certain carbon sources

Abstract When a cellobiose-grown inoculum of Clostridium thermocellum was transferred to either glucose or fructose as the sole carbon sourcem growth occurred only after a long lag of 180–200 h. We established that sugar uptake and phosphorylation were not limiting growth nor was the lag period the time take for a physiological adaptation process or for the growth of a mutant carried over in the cellibiose-grown incoculum. It became apparent that a mutation was occuring during the lag period in response to the selection pressure exerted by the presence of glucose or fructose as the sole carbon source. Once growth occurred on glucose and fructose, the cells could be transferred to cellobiose and back to glucose or fructose without exhibiting the long lag period. The change was stable over several transfers in the respective sugars.     

Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source

Plant roots secrete a complex polysaccharide mucilage that may provide a significant source of carbon for microbes that colonize the rhizosphere. High molecular weight mucilage was separated by high-pressure liquid chromatography gel filtration from low molecular weight components of pea root exudate. Purified pea root mucilage generally was similar in sugar and glycosidic linkage composition to mucilage from cowpea, wheat, rice, and maize, but appeared to contain an unusually high amount of material that was similar to arabinogalactan protein. Purified pea mucilage was used as the sole carbon source for growth of several pea rhizosphere bacteria, including Rhizobium leguminosarum 8401 and 4292, Burkholderia cepacia AMMD, and Pseudomonas fluorescens PRA25. These species grew on mucilage to cell densities of three- to 25-fold higher than controls with no added carbon source, with cell densities of 1 to 15% of those obtained on an equal weight of glucose. Micromolar concentrations of nod gene-inducing flavonoids specifically stimulated mucilage-dependent growth of R. leguminosarum 8401 to levels almost equaling the glucose controls. R. leguminosarum 8401 was able to hydrolyze p-nitrophenyl glycosides of various sugars and partially utilize a number of purified plant polysaccharides as sole carbon sources, indicating that R. leguminosarum 8401 can make an unexpected variety of carbohydrases, in accordance with its ability to extensively utilize pea root mucilage.     

Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::β-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Dark hexose metabolism by photoautotrophically and heterotrophically grown cells of the blue-green alga (Cyanobacterium) Nostoc sp. strain Mac.

Photoautotrophically grown cells of the blue-green alga (cyanobacterium) Nostoc sp. strain Mac assimilated and oxidized both glucose and fructose in the dark at different rates. The rate of fructose metabolism in these cells could be stimulated by casein hydrolysate, the effect being most pronounced at low sugar concentrations. This stimulation was not seen in cells grown heterotrophically in the dark, suggesting that it is a transitory phenomenon which disappears during the autotrophy-heterotrophy growth transition. The stimulation of fructose assimilation by casein hydrolysate was abolished by chloramphenicol or streptomycin, suggesting there are rate-limiting steps in protein biosynthesis in the dark that ultimately lead to inhibition of fructose uptake. Glucose metabolism did not show these phenomena, indicating there are differences in the metabolism of the two sugars.     

野生型和ospk1突变体水稻幼苗根对外源糖分的吸收和响应

植物根系如何响应环境因子变化是植物发育和营养吸收研究的重要科学问题。丙酮酸激酶OsPK1在根部的表达主要在根尖成熟区和根毛区,其表达水平变化有可能影响水稻对外源糖分的吸收。采用日本晴和水稻突变体ospk1,通过改变1/2 MS培养基中蔗糖含量,探索水稻幼苗对外源糖分的吸收和响应。通过GC-MS的方法检测了水稻幼苗叶片、叶鞘和根中蔗糖、葡萄糖、果糖和半乳糖的含量。发现根与培养基中糖分接触能明显提高幼苗中的糖含量。并且这些幼苗的根系长度大于那些不加蔗糖的培养基培养的幼苗,表明外源糖分被吸收后能促进根的伸长。OsPK1表达下调影响了糖代谢和外源糖分的吸收。半定量RT-PCR结果显示,幼苗根与糖分的直接接触明显上调根中OsPIP2;4,OsPIP2;5和OsTIP2;1三个水孔蛋白基因的表达。     

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Glucose, and not sucrose, is transported from wheat to wheat powdery mildew

The main host carbon energy source transferred from wheat leaves (Triticum aestivum L.) to wheat powdery mildew (Erysiphe graminis f.sp. tritici) has been investigated in three ways. When the uptake of sugars by isolated mycelial suspensions was examined, the uptake rate for glucose was considerably higher than that for a range of other solutes. Analysis by high-performance liquid chromatography of leaf and mycelial extracts following uptake of sugars into infected leaf pieces confirmed that sucrose was rapidly hydrolyzed in the leaf; no sucrose or fructose could be detected in mycelial extracts. Furthermore, studies of the uptake of asymmetrically labelled sucrose indicated that this sugar is cleaved prior to uptake by the pathogen. Thus several lines of evidence show that glucose, and not sucrose, is the major carbon energy source transferred from host to fungal mycelium. Received: 11 November 1998 / Accepted: 18 January 1999     

Evolutionary and ecophysiological significance of sugar utilization by the peat moss Sphagnum compactum (Sphagnaceae) and the common charophycean associates Cylindrocystis brebissonii and Mougeotia sp. (Zygnemataceae)

? Premise of the study: The goal of this study was to illuminate the evolutionary history and ecological importance of plant mixotrophy-the uptake and utilization of exogenous organic compounds. ? Methods: We quantitatively assessed the effect of sugar amendments on laboratory growth of Sphagnum compactum as a representative emergent peat moss and two species of ecologically associated zygnematalean algae, Cylindrocystis brebissonii and Mougeotia sp. ? Key results: Together with observations published elsewhere, our results suggest that under carbon or light limitation, the uptake of exogenous sugars by cells of charophycean algae and peat mosses may help these organisms maintain positive carbon balance. Utilization of 1% glucose by aquatic-grown algae helped to relieve dissolved inorganic carbon limitation, enhancing photoautotrophic growth by factors of 9.0 and 1.7, respectively. After an 8-wk growth period, amendments of 1% and 2% glucose enhanced air-grown moss biomass by 28 and 39 times, respectively, that of controls lacking sugar amendments. After 9 wk, 1% fructose enhanced biomass by 21 times, and 2% sucrose enhanced biomass by 31 times. ? Conclusion: Our results indicate that plant mixotrophy is an early-evolved trait. The results also indicate that quantitative differences in sugar utilization by bryophytes and charophycean algae correlate with relative investments in protective cell-wall polyphenolics measured in previous studies, suggesting that sugar utilization may subsidize the cost of producing phenolic wall compounds in bryophytes.