Purification and Partial Characterization of Genetically Engineered Thiol Protease Over-expressed in Escherichia Coli and Analysis of Priming Effects on Free Amino Acid Accumulation in Pinus Taeoa Seeds

The overall objective of an ongoing project is to determine the physiological and genetic mechanisms responsible for the invigoration of loblolly pine (Pinus taeda L.) seeds subjected to controlled water stress through solid matrix priming (SMP). It is hypothesized that the invigoration is due to a change in the sequence of events in the germinating seed where storage protein degradation and mobilization which normally commence after germination are stimulated to occur before radical emergence. The mobilization of protein reserves provides: osmotically active substances for osmotic adjustment leading to increased capacity of the embryo to grow and the components for synthesis of new proteins for the growing plant. It is also hypothesized that thiol protease (TP) is a major enzyme involved in the degradation of storage proteins when seeds are primed, as it is known to degrade reserve proteins, to be active during germination and to be up-regulated by water stress. As one effort toward the overall objective, the first chapter concerns TP antigen production to make anti-TP antibodies so that it becomes possible to further elucidate TP functions at the translation and post-translation levels cellularly and subcellularly. In addition, the over-expressed TP protein provides a way to efficiently purify the enzyme by immunoaffinity chromatography from loblolly pine. The second chapter, without direct relations to Chapter I but as another step toward the overall goal, concerns SMP effects on free amino acid accumulation and the relationships among free amino acid accumulation, TP activity increase and decrease of water potential

American ginseng modulates pancreatic beta cell activities

The mechanism of the beneficial effects of Panax quinquefolius (Xiyangshen, American ginseng) on diabetes is yet to be elucidated. Recent studies show that Panax quinquefolius increases insulin production and reduces the death of pancreatic beta cells. Mechanism studies indicate that Panax quinquefolius improves cell's immuno-reactivity and mitochondrial function through various factors. Clinical studies show that Panax quinquefolius improves postprandial glycemia in type 2 diabetic patients. Further studies to identify the component(s) of Panax quinquefolius linked with pancreatic islets/beta cells in vitro and in vivo are warranted for better understanding of the full effects of Panax quinquefolius

Cellulose synthase encoding polynucleotides and uses thereof

The invention relates to isolated polynucleotides encoding functional cellulose synthases and UDP-glucose binding domain thereof, transgenic plants and plant cells transformed with the polynucleotides. The invention further relates to methods of transforming plants and plant cells with cellulose synthase or UDP-encoding polynucleotides.https://digitalcommons.mtu.edu/patents/1113/thumbnail.jp

Cellulose synthase promoter and method for modifiying cellulose and lignin biosynthesis in plants

This invention relates to an isolated cellulose synthase promoter, methods for genetically altering cellulose and lignin biosynthesis, and to methods for improving strength properties of juvenile wood and fiber in trees. The invention further relates to methods for identifying regulatory elements in a cellulose synthase promoter and to methods for augmenting expression of polynucleotides operably linked to a cellulose synthase promoter.https://digitalcommons.mtu.edu/patents/1037/thumbnail.jp

Impact of tobramycin on the performance of microbial fuel cell

BACKGROUND: The release of antibiotics into aquatic environments has made the treatment of wastewater containing antibiotics a world-wide public health problem. The ability of microbial fuel cells (MFCs) to harvest electricity from organic waste and renewable biomass is attracting increased interest in wastewater treatment. In this paper we investigated the bioelectrochemical response of an electroactive mixed-culture biofilm in MFC to different tobramycin concentrations. RESULTS: The electroactive biofilms showed a high degree of robustness against tobramycin at the level of μg/L. The current generation responses of the biofilms were affected by the presence of tobramycin. The inhibition ratio of the MFC increased exponentially with the tobramycin concentrations in the range of 0.1-1.9 g/L. The bacterial communities of the biofilms vary with the concentrations of tobramycin, the equilibrium of which is critical for the stability of electroactive biofilms based-MFC. CONCLUSIONS: Experimental results demonstrate that the electroactive biofilm-based MFC is robust against antibiotics at the level of μg/L, but sensitive to changes in antibiotic concentration at the level of g/L. These results could provide significant information about the effects of antibiotics on the performance MFC as a waste-treatment technology

Enhancement of sugar yield by introducing a metabolic sink in sugarcane

Increasing sugar content is a primary objective of sugarcane improvement programs worldwide, since sugar accumulation in sugarcane is well below the theoretical physiological limits. Despite decades of conventional breeding and molecular manipulation, sugar content has remained stagnant in the new varieties for the past few decades. Recent experiments in genetic engineering have shown that sugar content in the transgenic sugarcanes was increased significantly (SugarBooster) by creating a metabolic sink through converting sucrose into slowly digested or inert products in the vacuoles of sugar storage cells. Technically, SugarBooster lines were created by introducing a gene for sucrose isomerase or fructosyl transferase into sugarcane to generate isomaltulose- or fructants-like products. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. Plant lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. The transgenic sugarcane line engineered to express a vacuole-targeted sucrose isomerase, which shows SugarBooster effects, was also found to accumulate sucrose to twice the concentration of the background genotype in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose concentrations declined over successive subcultures, but enhanced sucrose accumulation was stable. Detailed physiological characterization revealed multiple processes altered in the transgenic line in a direction consistent with enhanced sucrose accumulation. Striking differences from the control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose cleavage enzymes (particularly sucrose synthase (SuSy) breakage activity) and enhanced concentrations of symplastic hexose-6-phosphate and trehalose-6-phosphate in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate. Sucrose non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates SuSy breakage activity while down-regulating SPS in plants) was significantly lower in the transgenic line during the period of fastest sucrose accumulation. The SugarBooster effects elucidated from either transgenic plants or suspension-cultured cells provide hints at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane. Approaches are discussed for enhancing sugar content by manipulating multiple genes

Isomaltulose is actively metabolized in plant cells

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with V for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different V and V/K ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields

Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K(m) was 39.9 mM, the V(max) was 638 U mg(−1), and the K(cat)/K(m) was 1.79 × 10(4) M(−1) s(−1), compared to a K(m) of 76.0 mM, a V(max) of 423 U mg(−1), and a K(cat)/K(m) of 0.62 × 10(4) M(−1) s(−1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose

A xylem-specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress

Angiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant decrease in lignin in the cell walls of tension-stressed tissues. To investigate the molecular basis of this tree stress response, we cloned a full-length cellulose synthase (PtCesA) cDNA from developing xylem of aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts in various aspen tissues, and PtCesA gene promoter-β-glucuronidase (GUS) fusion analysis in transgenic tobacco, demonstrated conclusively that PtCesA expression is confined to developing xylem cells during normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in xylem and was induced in developing phloem fibers undergoing tension stress, but was turned off in tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for PtCesA in cellulose biosynthesis in both tension-stressed and normal tissues in aspen, and that the on/off control of PtCesA expression may be a part of a signaling mechanism triggering a stress-related compensatory deposition of cellulose and lignin that is crucial to growth and development in trees