Accumulation Of Mannitol Research Articles

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

ABSTRACTWe studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02 mg/g DW in comparison with the nontransformed roots (up to 0.687 mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.

Construction of an expression vector carrying gene coding mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli JM109 strain to transfer into maize

Maize (Zea mays L.) is one of the important cereal crops in the world, and the maize demand is increasing but the productivity and yield of maize have been affected by extreme adverse conditions, of tghose drought is one of the main factors reducing productivity due to inhibition of growth and decreasing photosynthesis in plants. Mannitol-1-phosphate dehydrogenase (mtlD) is an enzyme that catalyzes the conversion of fructose 2- and 6-phosphate to mannitol-1-phosphate in the photosynthesis pathway of plants. To produce maize containing mtlD gene to increase the height, fresh and dry weight, salt/drought tolerance in host plant based on accumulation of mannitol, we constructed an expression vector carrying the mtlD gene. The mtlD gene was isolated from E. coli JM109, cloned and sequenced in vector pJET1.2. This sequence was then optimized by OptimumGeneTM software to make it appropriate into plants. The modified gene was recombined into a mediate pRTRA vector containing 35S promoter and 35S terminator, the 35Spro::mtlD::35S cassette was construced into pCAMBIA1300 Ti-plasmid. The new recombinant vector was transformed into A. tumefaciens which could be used as a material for maize transformation. Citation: Le Bac Viet, Nguyen Thi Kim Lien, Nguyen Huy Hoang, Nong Van Hai, Huynh Thi Thu Hue, 2017. Construction of an expression vector carrying gene coding mannitol-1-phosphate dehydrogenase (MTLD) from Escherichia coli JM109 strain to transfer into maize. Tap chi Sinh hoc, 39(1): 61-67. DOI: 10.15625/0866-7160/v39n1.7073. *Corresponding author: hthue@igr.ac.vn Received 30 December 2015, accepted 20 March 2017

Transgenic peanut overexpressing mtlD gene confers enhanced salinity stress tolerance via mannitol accumulation and differential antioxidative responses

Globally, peanut is an important oilseed crop, which is cultivated under different agro-climatic zones. Soil salinity is one of the major constraints in peanut cultivation. Therefore, to understand the physio-biochemical mechanisms imparting salinity stress, four transgenic peanut lines (cv. GG20) already developed and confirmed by our lab, having bacterial mannitol dehydrogenase gene (mtlD), were subjected to different levels of salinity stresses (1, 2 and 3 dS m−1) in pots under containment facility. Further, these lines were also characterized for various physio-biochemical parameters at flowering, pegging and pod formation stages. All the transgenic lines recorded significantly higher mannitol dehydrogenase (MTD) activity and mannitol accumulation than the wild type (WT). Under salinity stress, significantly higher levels of superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase activities, while significantly lower levels of H2O2 and malondialdehyde contents, were recorded in the transgenics compared to WT. Similarly, significantly higher ascorbic acid and relative water content (RWC) were recorded in transgenic lines. The MTD activity showed positive correlation with various antioxidant enzymes, growth parameters and RWC, while negative correlation was recorded with H2O2 and malondialdehyde content at most of the plant growth stages. The mtlD transgenic peanut lines under pot conditions were found maintaining lower oxidative injuries, indicating amelioration of salinity-induced oxidative stress by enhanced protection mechanisms via mannitol accumulation and antioxidative responses. The best lines identified (MTD1 and MTD4) may be used further as pre-breeding source for imparting salinity stress tolerance in peanut. Besides, these lines may also be tested under open-field trials for release as salt-tolerant variety.

Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in Delphinium × belladonna cv. Bellamosum.

The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

Effect of osmotic stress on osmolyte accumulation and ammonia assimilating enzymes in chickpea cultivars

An investigation was conducted to evaluate the effect of osmotic stress on four chickpea cultivars, viz., two drought tolerant (ICC 4958 and Vijay), and two drought susceptible (JG-216 and PG 1808) subjected to mild (−0.4 MPa) and severe (−1.2 MPa) osmotic stress induced by PEG-6000. The activity of ammonia assimilation enzymes, viz., nitrate reductase (NR) glutamine synthetase (GS), glutamate synthase (GOGAT) decreased, while activity of proline synthesizing enzyme Δ1 pyrroline-5-carboxlate synthetase (P5CS), and contents of proline, glycine-betaine and mannitol increased significantly in both drought tolerant and susceptible chickpea cultivars under mild and severe osmotic stress conditions. The extent of increase was higher in tolerant than susceptible cultivars. ICC-4958 recorded higher accumulation of osmolytes, viz., proline, glycine-betaine, mannitol and P5CS activity with less decrease in the activity of NR, GS, and GOGAT.

Identification of mannitol as compatible solute in Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10mM) and 300mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol.

In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

Rapid cold hardening of Thrips palmi (Thysanoptera: Thripidae).

Cold tolerance of the palm thrips, Thrips palmi Karny, was investigated to predict its survival in field during winter. Supercooling points of T. palmi were varied among the developmental stages and ranged from -26.4 to -18.4°C. However, the cold injuries occurred above supercooling points in terms of higher mortality. The exposure to subzero temperatures (-5° to -15°C) resulted in significant mortalities to all developmental stages of T. palmi. A preexposure to a low temperature (4°C) for 7 h significantly increased the cold tolerance of all stages of T. palmi with respect to survival at -10°C and supercooling capacity. The rapid cold hardening (RCH) was dependent on the duration of the preexposure period at 4°C in adult stage. Polyol and sugar analysis using an high-performance liquid chromatography analysis showed that 4°C preexposure caused accumulation of glycerol, trehalose, mannitol, and mannose in the adults. The increase in trehalose levels was more significant than the others. This study suggests that all stages of T. palmi are able to become cold-hardy by RCH, in which several polyols and sugars may play crucial roles as cryoprotectants.

Homeologous genes involved in mannitol synthesis reveal unequal contributions in response to abiotic stress in Coffea arabica.

Polyploid plants can exhibit transcriptional modulation in homeologous genes in response to abiotic stresses. Coffea arabica, an allotetraploid, accounts for 75% of the world's coffee production. Extreme temperatures, salinity and drought limit crop productivity, which includes coffee plants. Mannitol is known to be involved in abiotic stress tolerance in higher plants. This study aimed to investigate the transcriptional responses of genes involved in mannitol biosynthesis and catabolism in C. arabica leaves under water deficit, salt stress and high temperature. Mannitol concentration was significantly increased in leaves of plants under drought and salinity, but reduced by heat stress. Fructose content followed the level of mannitol only in heat-stressed plants, suggesting the partitioning of the former into other metabolites during drought and salt stress conditions. Transcripts of the key enzymes involved in mannitol biosynthesis, CaM6PR, CaPMI and CaMTD, were modulated in distinct ways depending on the abiotic stress. Our data suggest that changes in mannitol accumulation during drought and salt stress in leaves of C. arabica are due, at least in part, to the increased expression of the key genes involved in mannitol biosynthesis. In addition, the homeologs of the Coffea canephora subgenome did not present the same pattern of overall transcriptional response, indicating differential regulation of these genes by the same stimulus. In this way, this study adds new information on the differential expression of C. arabica homeologous genes under adverse environmental conditions showing that abiotic stresses can influence the homeologous gene regulation pattern, in this case, mainly on those involved in mannitol pathway.

Diurnal Regulation of Leaf Water Status in High- and Low-Mannitol Olive Cultivars.

The role of mannitol and malic acid in the regulation of diurnal leaf water relations was investigated in ‘Biancolilla’ (high-mannitol) and ‘Cerasuola’ (low-mannitol) olive trees. Photosynthetic photon flux density (PPFD), vapor pressure deficit (VPD), stomatal conductance (gs), transpiration rate (T), relative water content (RWC), mannitol and malic acid were measured in ‘Biancolilla’ and ‘Cerasuola’ leaves during a dry and hot day of summer in Sicily. In general, leaves of ‘Biancolilla’ trees exhibited greater mannitol content, higher gs and T, but lower RWC than leaves of ‘Cerasuola’ trees. Differences in gs and T between the two cultivars were evident mainly in mid to late morning. ‘Biancolilla’ leaves accumulated mannitol at midday and again late in the evening. Stomatal responses to VPD were RWC dependent, and limited somewhat T, only in ‘Biancolilla’. Mannitol was directly related to RWC, and may play an osmotic role, in ‘Biancolilla’ leaves, whereas ‘Cerasuola’ leaves remained well hydrated by just transpiring less and regardless of mannitol. A day-time accumulation and night-time utilization of mannitol in ‘Biancolilla’ leaves is proposed as an efficient mechanism to regulate water status and growth.

Three Mitogen-Activated Protein Kinases Required for Cell Wall Integrity Contribute Greatly to Biocontrol Potential of a Fungal Entomopathogen

Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP) kinases constituting cell wall integrity (CWI) pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG) pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

Mannitol is essential for the development of stress-resistant ascospores in Neosartorya fischeri (Aspergillus fischeri)

The polyol mannitol is one of the main compatible solutes in Neosartorya fischeri and accumulates in conidia and ascospores. Here, it is shown that biosynthesis of mannitol in N. fischeri mainly depends on mannitol 1-phosphate dehydrogenase (MpdA). Reporter studies and qPCR analysis demonstrated that mpdA is moderately expressed in vegetative hyphae and conidiophores, while it is highly expressed during development of ascospores. Deletion of mpdA reduced mannitol in whole cultures as much as 85% of the wild type, while trehalose levels had increased more than 4-fold. Decreased mannitol accumulation had no effect on mycelial growth irrespective of heat- or oxidative stress. Notably, conidia of the ΔmpdA strain had higher mannitol and lower trehalose levels. They were more sensitive to heat stress. The most distinct phenotype of mpdA deletion was the absence of full development of ascospores. Formation of cleistothecia, and asci was not affected. The ascus cell wall, however, did not dissolve and asci contained incompletely formed or aborted ascospores. Addition of the Mpd inhibitor nitrophenide to the wild type strain also resulted in disturbed ascospore formation. Taken together, these results show that mannitol has a role in sexual development of N. fischeri and in stress resistance of conidia.

Iodine contributes to osmotic acclimatisation in the kelp Laminaria digitata (Phaeophyceae).

Iodide (I⁻) retained by the brown macroalga Laminaria digitata at millimolar levels, possesses antioxidant activities, but the wider physiological significance of its accumulation remains poorly understood. In its natural habitat in the lower intertidal, L. digitata experiences salinity changes and osmotic homeostasis is achieved by regulating the organic osmolyte mannitol. However, I⁻ may also holds an osmotic function. Here, impacts of hypo- and hypersaline conditions on I⁻ release from, and accumulation by, L. digitata were assessed. Additionally, mannitol accumulation was determined at high salinities, and physiological responses to externally elevated iodine concentrations and salinities were characterised by chl a fluorometry. Net I⁻ release rates increased with decreasing salinity. I⁻ was accumulated at normal (35 S A) and high salinities (50 S A); this coincided with enhanced rETRmax and qP causing pronounced photoprotection capabilities via NPQ. At 50 S A elevated tissue iodine levels impeded the well-established response of mannitol accumulation and prevented photoinhibition. Contrarily, low tissue iodine levels limited photoprotection capabilities and resulted in photoinhibition at 50 S A, even though mannitol was accumulated. The results indicate a, so far, undescribed osmotic function of I⁻ in L. digitata and, thus, multifunctional principles of this halogen in kelps. The osmotic function of mannitol may have been substituted by that of I⁻ under hypersaline conditions, suggesting a complementary role of inorganic and organic solutes under salinity stress. This study also provides first evidence that iodine accumulation in L. digitata positively affects photo-physiology.

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium

d-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1gmannitolL−1 and a production rate of 0.15gmannitolL−1day−1. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria.

Comparative metabolic profiling revealed limitations in xylose‐fermenting yeast during co‐fermentation of glucose and xylose in the presence of inhibitors

During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose-derived inhibitors, which disrupt the efficiency of hexose and pentose co-fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH-ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and g-aminobutyric acid (GABA) suggested that strain 424A (LNH-ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co-fermentation than glucose fermentation was observed in strain 424A (LNH-ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH-ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH-ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH-ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors.

Gas-Chromatography Mass-Spectrometry (GC-MS) Based Metabolite Profiling Reveals Mannitol as a Major Storage Carbohydrate in the Coccolithophorid Alga Emiliania huxleyi.

Algae are divergent organisms having a wide variety of evolutional histories. Although most of them share photosynthetic activity, their pathways of primary carbon metabolism are rather diverse among species. Here we developed a method for gas chromatography-mass spectroscopy (GC-MS) based metabolite profiling for the coccolithophorid alga Emiliania huxleyi, which is one of the most abundant microalgae in the ocean, in order to gain an overview of the pathway of primary metabolism within this alga. Following method optimization, twenty-six metabolites could be detected by this method. Whilst most proteogenic amino acids were detected, no peaks corresponding to malate and fumarate were found. The metabolite profile of E. huxleyi was, however, characterized by a prominent accumulation of mannitol reaching in excess of 14 nmol 106 cells−1. Similarly, the accumulation of the 13C label during short term H13CO3− feeding revealed a massive redistribution of label into mannitol as well as rapid but saturating label accumulation into glucose and several amino acids including aspartate, glycine and serine. These results provide support to previous work suggesting that this species adopts C3 photosynthesis and that mannitol functions as a carbon store in E. huxleyi.

Marker-free Transgenic Tomato with Engineered Mannitol Accumulation Confers Tolerance to Multiple Abiotic Stresses

The co-transformation system using mixture of two Agrobacterium cultures, one harbouring bacterial mannitol-1- phosphate dehydrogenase (mtlD) as gene of interest, whereas the other harboring the neomycin phosphotransferase (nptII) marker gene for selection, was successfully used to develop selectable marker-free transgenics of tomato with engineered mannitol accumulation, to improve tolerance against multiple abiotic stresses. We have achieved high co-transformation frequency (up to 24%), and segregation frequency (up to 22-24%) for obtaining marker-free transgenics. Marker-free transgenic tomato plants resulted in mannitol accumulation, as evident by HPLC analysis, that leads to substantial protection of the vigour of the plants under multiple abiotic stress conditions, such as high salinity (up to 200 mM NaCl), drought (up to 15% PEG), and heavy-metal toxicity (up to 2 mM CdCl2). Under stress, transgenics performed well in comparison to wild type (WT), with high percentage of seed germination and seedling growth both in vitro and in vivo. They also performed better than WT, in terms of high chlorophyll content and other photosynthetic parameters such as Fv/Fm ratio, Electron Transport Rate (ETR), photosynthetic yield, and low Non Photochemical Quenching (NPQ). Thus, high mannitol level not only increases the nutritional value of the fruit, but also improves tolerance of transgenic tomato plants by increasing its physiological and photosynthetic efficiency, suggesting that carbohydrate alterations produced by mannitol biosynthesis may be linked to the stress response.

POSSIBLE ROLE OF MANNITOL AS AN OXYGEN RADICAL SCAVENGER IN OLIVE

POSSIBLE ROLE OF MANNITOL AS AN OXYGEN RADICAL SCAVENGER IN OLIVE

Biotechnological valorization of low-cost sugar-based media for bacteriocin production by Leuconostoc mesenteroides E131

The aim of the present study was to evaluate the suitability of low-cost carbon sources for bacteriocin production by Leuconostoc mesenteroides strain E131. For this purpose, inexpensive sugars derived from a sugar refinery plant (glucose, fructose and sucrose) as well as waste molasses were utilized as carbon sources in submerged shake-flask experiments and the kinetic response of the microorganism was evaluated. Interestingly, in the case of molasses, non-negligible decolorization-detoxification (up to ∼27%) of the residue was performed together with the production of bacteriocin. In all instances the initial concentration of sugars employed was adjusted at 20 and 30 g/L, therefore the effect of both the nature and the initial quantity of sugar upon the growth of the microorganism was assessed. All media proved to be suitable for both biomass and bacteriocin production by L. mesenteroides, whereas variable quantities of lactate, acetate and ethanol were detected into the medium. Employment of fructose, sucrose or molasses as carbon sources resulted in the accumulation of mannitol (in some cases in significant quantities) into the medium; remarkable portion thus of the available or released fructose acted as electron acceptor instead of carbon source by the microorganism. The highest bacteriocin production achieved (=640 AU/mL) was obtained when initial glucose at 30 g/L was used as substrate. Finally, utilization of waste molasses as carbon source by L. mesenteroides resulted in satisfactory bacteriocin production (up to 320 AU/mL) besides the decolorization of the residue.

Enhanced salt stress tolerance in transgenic potato plants (Solanum tuberosum L.) expressing a bacterial mtlD gene

Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l−1 kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.