Sucrose Depletion Research Articles

Senescence metabolomics of <i>Nicotiana tabacum</i> L. VBI-0 heterotrophic suspension cultures

BACKGROUND: Heterotrophic cell cultures are widely used as a model in plant biology. During a culture cycle the composition of the medium changes: the sucrose and other substrates are depleted, metabolism products are accumulated and the density increases. Finally, arrest of a growth is followed by cell death in a short time. These processes are accompanied with physiological alterations, corresponding to senescence. AIM: To resolve metabolic features of tobacco cells in growing and stationary senescent suspension cultures VBI-0. MATERIALS AND METHODS: Nicotiana tabacum VBI-0 cells were cultured in suspension MS medium supplied with 3% sucrose. Cells were sampled at 7th day, during intensive growth, and at 28th day, when the culture was in the stationary phase. The GC-MS method was used to profile the metabolites. RESULTS: Sucrose depletion in media caused starvation of heterotrophic tobacco cell culture and was associated with a decrease in the accumulation of free amino acids. At the same time, the level of pentoses and complex sugars, including sucrose, increased, while the levels of glucose and fructose were not changed significantly and levels of hexose phosphates decreased. During culture senescence cells showed higher levels of accumulation of malate, pyruvate and some other carboxylates. CONCLUSIONS: The metabolomic data indicate that culture senescence was associated with a drop in amino acids metabolism, a decrease in the activity of the upper part of glycolysis, and the accumulation of complex sugars, pentoses and carboxylates.

Change in Sucrose Cleavage Pattern and Rapid Starch Accumulation Govern Lily Shoot-to-Bulblet Transition in vitro.

In bulb crops, bulbing is a key progress in micropropagation and is the feature that most distinguishes bulbous crops from other plants. Generally, bulbing involves a shoot-to-bulblet transition; however, the underlying mechanism remains elusive. We explored this process by tracking the shoot-to-bulblet transition under different culture conditions. Rapid starch accumulation occurred at 15 days after transplanting (DAT) in the bulblet-inducing treatments as confirmed via histological observations and the significant elevation of starch synthesis related-gene transcription, including LohAGPS, LohAGPL, LohGBSS, LohSS, and LohSBE. However, for shoots that did not transition to bulblets and maintained the shoot status, much higher soluble sugars were detected. Interestingly, we observed a clear shift from invertase-catalyzed to sucrose synthase-catalyzed sucrose cleavage pattern based on the differential expression of LohCWIN and LohSuSy during the key transition stage (prior to and after bulbing at 0–15 DAT). Shoots that transitioned into bulblets showed significantly higher LohSuSy expression, especially LohSuSy4 expression, than shoots that did not transition. A symplastic phloem unloading pathway at the bulblet emergence stage (15 DAT) was verified via the 6(5)-carboxyfluorescein diacetate fluorescent tracer. We propose that starch is the fundamental compound in the shoot-to-bulblet transition and that starch synthesis is likely triggered by the switch from apoplastic to symplastic sucrose unloading, which may be related to sucrose depletion. Furthermore, this study is the first to provide a complete inventory of the genes involved in starch metabolism based on our transcriptome data. Two of these genes, LohAGPS1.2b and LohSSIIId, were verified by rapid amplification of cDNA ends cloning, and these data will provide additional support for Lilium research since whole genome is currently lacking.

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Factors Affecting Bulblet Growth of Lilium sp. - Tracking Ontogenic Development and Bulb Production in vitro

Poor growth of bulblet and ontogenic development are the major problems of adventitious bulb production of lilies in vitro through explant culture. Ontogenic development from the juvenile phase to the adult vegetative phase strongly correlates with bulblet growth of lily, and it is important to understand how lily bulblets grow in vitro. This study was at aimed to determining an effective in vitro culture process for lily to track bulblet growth, ontogenic development, and bulb production. In the results, bulblet performance was higher with the increase in the amount of medium; however, bulblet size was not satisfactory and was in the juvenile phase under an inducing condition. The threshold weight at which 100% ontogenic development was about 300 mg. Avoiding depletion of sucrose in the media is the key to achieving desired bulblet growth and ontogenic development in vitro and indicates growth could be sustained by subculture. The circumference of bulblets increases with its weight after subcultured in vitro. Therefore, a threshold circumference of about 3.2 cm in proportion to bulblet fresh weight of 300 - 350 mg may be an indicator of ontogenic development.
 Plant Tissue Cult. & Biotech. 30(1): 1-13, 2020 (June)

Entrapment of glutaraldehyde‐crosslinked cells from Aspergillus oryzaeIPT‐301 in calcium alginate for high transfructosylation activity

AbstractBACKGROUNDFructooligosaccharides (FOS) are sweeteners produced through the use of enzymes called fructosyltransferases (FTases) (EC 2.4.1.9). However, FTase reuse is meagre and the cost of the enzyme production is high, making FOS production economically unfeasible. In order to address these issues, this study aims to immobilize cells from Aspergillus oryzae IPT‐301, containing FTase enzyme previously glutaraldehyde‐crosslinked, by means of entrapment in calcium alginate beads. The conditions to obtain the particles, such as alginate type, mycelium mechanical dispersion, degree of crosslinking, size of the beads, and their reflections on transfructosylation activity (AT) were evaluated previously.RESULTSThe highest AT used low‐viscosity alginate solution (4.0% w/w) crosslinked with calcium chloride (0.2 mol L−1) and beads of mean diameter 2.15 mm. The entrapment conditions were complemented by conducting the reaction under different temperatures, pH values, substrate concentrations and by reusing the beads. The highest transfructosylation activities were obtained under reaction conditions of 50 °C, pH 5.5 and substrate concentration 400 g L−1.CONCLUSIONThe AT levels found with the glutaraldehyde‐crosslinked and entrapped cells in calcium alginate were ≈70% higher than the ones obtained with free cells, not glutaraldehyde‐crosslinked. Moreover, the immobilized cells (crosslinked+entrapped) retained their AT up to the third cycle of total sucrose depletion in the reaction medium, attesting to the potential use of the beads in continuous FOS production. © 2020 Society of Chemical Industry

1H NMR metabolomic approach reveals chlorogenic acid as a response of sugarcane induced by exposure to Diatraea saccharalis

Sugarcane (Saccharum officinarum) has been considered one of the most efficient energy crops, but its production yield is sensitive to outbreaks of pest insects, especially the sugarcane borer Diatraea saccharalis. Genetic breeding programs and biotechnology projects have been developed to decode the defense mechanisms of sugarcane against herbivorous insect attacks, and the develop plague-resistant plants. We performed metabolic profile analysis of the SP791011 sugarcane variety’s response to Diatraea saccharalis herbivory, using Nuclear Magnetic Resonance (NMR) spectroscopy of organic leaf extracts. The leaf response of SP791011 to D. saccharalis resulted in depletion of choline, alanine, sucrose, glutamate, trigonelline, and isomers (E)-aconitate, (Z)-aconitate, and higher expression of chlorogenic acid and other caffeic acid conjugates in sugarcane leaves. The increase in chlorogenic acid suggests the shikimic acid pathway was induced by D. saccharalis herbivory, increasing the biosynthesis of phenylpropanoids such as chlorogenic acid in the sugarcane leaves. In addition to the herbivory test, we performed an in vivo biological assay by adding chlorogenic acid to an artificial diet to D. saccharalis caterpillars. This assay demonstrated a decrease in the development time of the pupae compared with pupae from caterpillars raised under normal diet. However, deformations in moth wings fed with chlorogenic acid were observed for three concentrations tested (0.05 mg/mL, 0.5 mg/mL and 5 mg/mL) during the in vivo bioassay. Chlorogenic acid may be considered a natural biopesticide and its production could be induced to develop more resistant sugarcane varieties against D. saccharalis.

Cryptochrome-mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis.

Both phototropins (phot1 and phot2) and cryptochromes (cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids (GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high‐intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3 (nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose‐containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3. Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.

Grape berry position affects the diurnal dynamics of its metabolic profile.

Solar irradiance and air temperature are characterized by dramatic circadian fluctuations and are known to significantly modulate fruit composition. To date, it remains unclear whether the abrupt, yet predictive, diurnal changes in radiation and temperature prompt direct metabolic turn-over in the fruit. We assessed the role of fruit insolation, air temperature, and source-tissue CO2 assimilation in the diurnal compositional changes in ripening grape berries. This was performed by comparing the diurnal changes in metabolite profiles of berries positioned such that they experienced (a) contrasting diurnal solar irradiance patterns, and (b) similar irradiance but contrasting diurnal CO2 assimilation patterns of adjacent leaves. Grape carbon levels increased during the morning and decreased thereafter. Sucrose levels decreased throughout the day and were correlated with air temperature, but not with the diurnal pattern of leaf CO2 assimilation. Tight correlation between sucrose and glucose-6-phosphate indicated the involvement of photorespiration/glycolysis in sucrose depletion. Amino acids, polyamines, and phenylpropanoids fluctuated diurnally, and were highly responsive to the diurnal insolation pattern of the fruit. Our results fill the knowledge gap regarding the circadian pattern of source-sink assimilate-translocation in grapevine. In addition, they suggest that short-term direct solar exposure of the fruit impacts both its diurnal and nocturnal metabolism.

TAT1 and TAT2 tyrosine aminotransferases have both distinct and shared functions in tyrosine metabolism and degradation in Arabidopsis thaliana

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower 13C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

Sucrose affects the developmental transition of rhizomes in Oryza longistaminata

Oryza longistaminata, the African wild rice, can propagate vegetatively through rhizomes. Rhizomes elongate horizontally underground as sink organs, however, they undergo a developmental transition that shifts their growth to the surface of the ground to become aerial stems. This particular stage is essential for the establishment of new ramets. While several determinants such as abiotic stimuli and plant hormones have been reported as key factors effecting developmental transition in aerial stem, the cause of this phenomenon in rhizome remains elusive. This study shows that depletion of nutrients, particularly sucrose, is the key stimulus that induces the developmental transition in rhizomes, as indicated by the gradient of sugars from the base to the tip of the rhizome. Sugar treatments revealed that sucrose specifically represses the developmental transition from rhizome to aerial stem by inhibiting the expression of sugar metabolism and hormone synthesis genes at the bending point. Sucrose depletion affected several factors contributing to the developmental transition of rhizome including signal transduction, transcriptional regulation and plant hormone balance.

Depletion of sucrose induces changes in the tip growth mechanism of tobacco pollen tubes.

Pollen tubes are rapidly growing, photosynthetically inactive cells that need high rates of energy to support growth. Energy can derive from internal and external storage sources. The lack of carbon sources can cause various problems during pollen tube growth, which in turn could affect the reproduction of plants. We analysed the effects of energy deficiency on the development of Nicotiana tabacum pollen tubes by replacing sucrose with glycerol in the growth medium. We focused on cell growth and related processes, such as metabolite composition and cell wall synthesis. We found that the lack of sucrose affects pollen germination and pollen tube length during a specific growth period. Both sugar metabolism and ATP concentration were affected by sucrose shortage when pollen tubes were grown in glycerol-based media; this was related to decreases in the concentrations of glucose, fructose and UDP-glucose. The intracellular pH and ROS levels also showed a different distribution in pollen tubes grown in sucrose-depleted media. Changes were also observed at the cell wall level, particularly in the content and distribution of two enzymes related to cell wall synthesis (sucrose synthase and callose synthase). Furthermore, both callose and newly secreted cell wall material (mainly pectins) showed an altered distribution corresponding to the lack of oscillatory growth in pollen tubes. Growth in glycerol-based media also temporarily affected the movement of generative cells and, in parallel, the deposition of callose plugs. Pollen tubes represent an ideal model system for studying metabolic pathways during the growth of plant cells. In our study, we found evidence that glycerol, a less energetic source for cell growth than sucrose, causes critical changes in cell wall deposition. The evidence that different aspects of pollen tube growth are affected is an indication that pollen tubes adapt to metabolic stress.

Effect of nutrients starvation in the cell cycle synchronization of cell suspension cultures of Jatropha curcas L.

The knowledge about the processes that control cell cycle activity in J. curcas has not yet been addressed. Cell suspension cultures offer an attractive alternative to carry out this kind of studies in a simplified biological system. Nevertheless, the cell suspensions exhibit a high degree of heterogeneity concerning cell cycle distribution; therefore, synchronization methods must be previously established for this plant species. In the present research, cell suspensions of J. curcas were used to assess phosphorus, nitrogen or sucrose starvation as cell cycle synchronization approaches. Cell suspension cultures were established from the endosperm of seeds using a method for the enzymatic breakdown, without a previous callogenesis stage. The cell suspensions reached a maximum growth rate of 0.15 d−1 and a doubling time of 4.8 d. Microscopy analyses showed amyloplasts in some of the cells; however, oil and protein bodies were not observed. Regarding cell cycle synchronization, the results indicated that phosphorus or nitrogen starvation did not affect the cell-cycle arrest in the G0/G1 phase. However, the sucrose depletion for three days induced an accumulation of 67% of the cells in the G0/G1 phase. This effect was reversed with the addition of a carbon source. Moreover, these culture conditions generated a population of cells (approx. 5%) with a higher DNA content (3n > 6n), which was interpreted as the activation of an endoreplication process. Our findings suggest that nutrient starvation can affect in a differential way the cell synchronization process.

Dextran Utilization During Its Synthesis by Weissella cibaria RBA12 Can Be Overcome by Fed-Batch Fermentation in a Bioreactor.

Weissella cibaria RBA12 produced a maximum of 9mg/ml dextran (with 90% efficiency) using shake flask culture under the optimized concentration of medium components viz. 2% (w/v) of each sucrose, yeast extract, and K2HPO4 after incubation at optimized conditions of 20°C and 180rpm for 24h. The optimized medium and conditions were used for scale-up of dextran production from Weissella cibaria RBA12 in 2.5-l working volume under batch fermentation in a bioreactor that yielded a maximum of 9.3mg/ml dextran (with 93% efficiency) at 14h. After 14h, dextran produced was utilized by the bacterium till 18h in its stationary phase under sucrose depleted conditions. Dextran utilization was further studied by fed-batch fermentation using sucrose feed. Dextran on production under fed-batch fermentation in bioreactor gave 35.8mg/ml after 32h. In fed-batch mode, there was no decrease in dextran concentration as observed in the batch mode. This showed that the utilization of dextran by Weissella cibaria RBA12 is initiated when there is sucrose depletion and therefore the presence of sucrose can possibly overcome the dextran hydrolysis. This is the first report of utilization of dextran, post-sucrose depletion by Weissella sp. studied in bioreactor.

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937–946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-d-Glcp-catechol and α-d-Glcp-(1→4)-α-d-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-d-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-d-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.

Effect of organochlorine pesticides exposure on the maize root metabolome assessed using high-resolution magic-angle spinning 1H NMR spectroscopy

1H-HRMAS NMR-based metabolomics was used to better understand the toxic effects on maize root tips of organochlorine pesticides (OCPs), namely lindane (γHCH) and chlordecone (CLD). Maize seedlings were exposed to 2.5 μM γHCH (mimicking basic environmental contaminations) for 7 days and compared to 2.5 μM CLD and 25 μM γHCH for 7 days (mimicking hot spot contaminations). The 1H-HRMAS NMR-based metabolomic profiles provided details of the changes in carbohydrates, amino acids, tricarboxylic acid (TCA) cycle intermediates and fatty acids with a significant separation between the control and OCP-exposed root tips. First of all, alterations in the balance between glycolysis/gluconeogenesis were observed with sucrose depletion and with dose-dependent fluctuations in glucose content. Secondly, observations indicated that OCPs might inactivate the TCA cycle, with sizeable succinate and fumarate depletion. Thirdly, disturbances in the amino acid composition (GABA, glutamine/glutamate, asparagine, isoleucine) reflected a new distribution of internal nitrogen compounds under OCP stress. Finally, OCP exposure caused an increase in fatty acid content, concomitant with a marked rise in oxidized fatty acids which could indicate failures in cell integrity and vitality. Moreover, the accumulation of asparagine and oxidized fatty acids with the induction of LOX3 transcription levels under OCP exposure highlighted an induction of protein and lipid catabolism. The overall data indicated that the effect of OCPs on primary metabolism could have broader physiological consequences on root development. Therefore, 1H-HRMAS NMR metabolomics is a sensitive tool for understanding molecular disturbances under OCP exposure and can be used to perform a rapid assessment of phytotoxicity.

Changes in soymilk during fermentation with kefir culture: oligosaccharides hydrolysis and isoflavone aglycone production

The objective of this study was to evaluate the changes in oligosaccharides and isoflavone aglycone content in soymilk during fermentation with commercial kefir culture. Soymilk was fermented with kefir culture at 25 °C for 30 h. The counts of lactic acid bacteria, Lactococcus lactis, Leuconostoc sp and yeasts; measurements of pH, acidity, α-galactosidase and β-glucosidase activity, sugar and isoflavone contents were performed at the intervals of time. In the fermented soymilk, the lactic acid bacteria counts increased from 7.6 log to 9.1 CFU g−1, pH reached to 4.9 and lactic acid reached 0.34 g 100 g− 1. The α-galactosidase was produced (0.016 AU g−1) with 100% raffinose and 92% stachyose hydrolysis being observed after the depletion of galactose, glucose and sucrose. Kefir culture produced β-glucosidase (0.0164 AU g−1), resulting in 100% bioconversion of glycitin and daidzin and 89% bioconversion of genistin into the corresponding aglycones. The fermented soymilk presented 1.67 μmol g−1 of daidzein, 0.28 μmol g−1 of glicitein and 1.67 μmol g −1 of genistein.

Time-dependent Changes in the Longitudinal Sugar and Respiratory Profiles of Asparagus Spears During Storage at 0 °C

The rate of respiration and the concentrations of sucrose, glucose, and fructose were measured along the length of intact asparagus (Asparagus officinalis cv. Jersey Giant) spears during storage at 0 °C. Carbon dioxide production by each of five sections along the spear was initially high but underwent a rapid and extensive decline within the first 24 hours after harvest with the rate of decline slowing thereafter. The respiration rate was highest at the tip (Section 1), decreasing as the distance from the tip increased (Sections 2 through 5 with Section 5 being more basal). Initially, the respiration rate of the tip was approximately four times that of the base, but after 23 days at 0 °C, the respiration rate of the tip was only twice that of the base. Sugar levels were measured in Sections 1 through 4. Sugar levels declined with time, but increased, unlike respiration, with distance from the tip. Sucrose underwent a rapid decline within the first 24 hours of storage in the tip and Sections 3 and 4. Sucrose depletion was most extensive in the tip, reaching more than 95% by Day 23. Glucose underwent the most rapid decline in Section 2. The relatively higher rate of glucose depletion in Section 2, the zone of rapid cell elongation, may have been to support a relatively higher rate of cell wall biosynthesis in this section. For the first day after harvest, sugar depletion far outstripped hexose equivalents respired as CO2. Afterward, however, the rate of respiration (as hexose equivalents) was similar to the rate of sugar depletion for all sections except the most basipetal, which lost carbohydrate faster than could be accounted for by respired CO2. The data suggest that hexoses were exported from more basipetal tissues to support the metabolic activity of more acropetal sections.

Differential Expression of Genes Encoding Acid Invertases in Multiple Shoots of Bamboo in Response to Various Phytohormones and Environmental Factors

The promoter regions of two cell wall invertase genes, Boβfruct1 and Boβfruct2, and a vacuolar invertase gene, Boβfruct3, in Bambusa oldhamii were cloned, and putative regulatory cis-elements were identified. The expression of these three genes in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The two cell wall invertase genes were upregulated by indole-3-acetic acid and cytokinins but responded differently to other phytohormones and different temperatures. Boβfruct1 was also upregulated by sucrose and glucose. In contrast, the Boβfruct2 expression was induced by the depletion of sucrose, and this induction could be suppressed by glucose and sucrose. The expression of Boβfruct3 was light-dependent; however, abscisic acid (ABA) could induce its expression in the dark. ABA and light exhibited an additive effect on the expression of Boβfruct3. Our results suggest that these three Boβfruct genes have individual roles in the adaption of the plant to environmental changes. Boβfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boβfruct3 might be one of the interacting nodes of the light and ABA signaling pathways.

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l−1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l−1) was achieved in media containing 150 g l−1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l−1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.

Effects of initial ammonium ion concentration on pullulan production by Aureobasidium pullulans and its modeling

Pullulan fermentations by Aureobasidium pullulans with various initial ammonium ion concentrations were evaluated in a 2-L bioreactor. The results demonstrated that A. pullulans produced highest pullulan (23.1 g/L) when the initial ammonium sulfate was 7 at 5 g/L. The purity of produced pullulan was 94.6%. Seven gram per liter of ammonium sulfate produced more biomass due to the higher level of nitrogen source, but the pullulan-degrading enzyme activity was detected after the depletion of sucrose, which reduced pullulan concentration. From the results of fed-batch fermentation, addition of 10 g/L of sucrose suppressed A. pullulans from producing pullulan-degrading enzyme. Additionally, the modified-Gompertz equation demonstrated its generality to fit all pullulan production, biomass production, and sucrose consumption curves at three ammonium sulfate levels. After incorporating the degrading factor, a re-modified-Gompertz equation was obtained that can adequately describe the decrease of pullulan at the late fermentation stage.