Robert D. Locy

Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.

Photosynthesis in Salt-Adapted Heterotrophic Tobacco Cells and Regenerated Plants.

Tobacco (Nicotiana tabacum L.) cells growing heterotrophically in the light on supplied sucrose (S0) have previously been adapted to grow in 428 mM NaCl (S25). Among the changes occurring in salinity-adapted cell cultures are (a) elevated levels of chlorophyll compared to unadapted cells; (b) decreased levels of starch; (c) alterations in chloroplast ultrastructure, including loss of starch grains, increased thylakoid membrane structure, and the presence of plastoglobules; and (d) increased rates of O2 evolution, CO2 fixation, and photophosphorylation relative to S0 cells. These latter changes apparently derive from the fact that thylakoid membranes in S25 cells contain higher levels of photosystem I- and II-associated proteins as well as thylakoid ATPase components. S25 chloroplasts contain immunologically detectable levels of ribulose-1,5-bisphosphate carboxylase/oxygenase, whereas S0 completely lack the enzyme. These changes taken together suggest that even in the presence of sucrose, S25 cells have acquired a significant degree of salt-tolerant photosynthetic competence. This salt-tolerant photoysynthetic capability manifests itself in plants backcrossed with normal plants for three generations. These plants contain chloroplasts that demonstrate in vitro more salt-tolerant CO2 fixation, O2 evolution, and photophosphorylation than do backcross progeny of plants regenerated from S0 cultures.

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO(3)(-)) in the growth medium. At NO(3)(-) concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO(3)(-), the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO(3)(-) uptake at low NO(3)(-), while GABA inhibited NO(3)(-) uptake at high NO(3)(-). Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.

Identification of a pyridoxine (pyridoxamine) 5′-phosphate oxidase from Arabidopsis thaliana

Pyridoxine (pyridoxamine) 5′‐phosphate oxidase (PPOX) catalyzes the oxidative conversion of pyridoxamine 5′‐phosphate (PMP) or pyridoxine 5′‐phosphate (PNP) to pyridoxal 5′‐phosphate (PLP). The At5g49970 gene of Arabidopsis thaliana shows homology to PPOXs from a number of organisms including the Saccharomyces cerevisiae PDX3 gene. A cDNA corresponding to putative A. thaliana PPOX (AtPPOX) was obtained using reverse transcriptase‐polymerase chain reaction and primers landing at the start and stop codons of At5g49970. The putative AtPPOX is 530 amino acid long and predicted to contain three distinct parts: a 64 amino acid long N‐terminal putative chloroplast transit peptide, followed by a long Yjef_N domain of unknown function and a C‐terminal Pyridox_oxidase domain. Recombinant proteins representing the C‐terminal domain of AtPPOX and AtPPOX without transit peptide were expressed in E. coli and showed PPOX enzyme activity. The PDX3 knockout yeast deficient in PPOX activity exhibited sensitivity to oxidative stress. Constructs of AtPPOX cDNA of different lengths complemented the PDX3 knockout yeast for oxidative stress. The role of the Yjef_N domain of AtPPOX was not determined, but it shows homology with a number of conserved hypothetical proteins of unknown function.

Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata)

Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

Purification and characterization of glutamate decarboxylase from cowpea

Abstract Glutamate decarboxylase (GAD) was purified 300-fold from green cowpea ( Vigna unguiculata L.) pods using a combination of PEG precipitation, DEAE cellulose chromatography, hydroxylapatite chromatography, and Q-resin chromatography. The partially purified preparation demonstrated 2 primary bands in SDS-polyacrylamide gel electrophoresis with up to 3 additional minor bands. Cowpea GAD has a pH optimum at between pH 5.5–6.0, and an apparent K M for glutamate of 3.2 mM at pH 5.8. Both crude GAD preparations and preparations partially purified through the hydroxylapatite step can be stimulated by Ca 2+ and calmodulin when assayed at pH 5.8. However, the purified enzyme does not show activation by Ca 2+ and/or calmodulin at pH 5.8 or at pH 7.0.

Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana

Abstract Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens–mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana.

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsisthaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciensby electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.

Growth of callus initiated from cucumber hypocotyls on galactose and galactose-containing oligosaccharides

Abstract Initiation and growth of cucumber hypocotyl callus on galactose, glucose, sucrose, melibiose, raffinose, stachyose, and sucrose + galactose (1 : 1, w/w) were examined. Abundant callus was produced on all of the above carbon sources. However, callus grew best on stachyose or raffinose after a substantial lag period (relative to growth on sucrose). Callus growth was similar on sucrose or galactose. α-Galactosidase activity was detected in hypocotyl explants and in calli grown on raffinose, melibiose, sucrose and galactose. Regardless of carbon source, cultured cells accumulated primarily sucrose, glucose and fructose internally. Free space sugars varied depending on carbon source and provided evidence of an extracellular location for α-galactosidase and invertase from cucumber cells.

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright