Search

The goals of this study were to express bovine lysozyme in tobacco and to purify the protein with a scaleable process to >90% homogeneity while retaining antimicrobial characteristics. Results showed that the enzyme was expressed at levels equivalent to 1−1.5% of total fraction 2 protein in each of five different transformant groups. The enzyme was subsequently purified to 93% homogeneity using an easily scaleable process while retaining high activity. It was concluded that tobacco was an excellent choice for expression of the recombinant protein and that the purification process developed in this study demonstrates methodology for isolation of high-value enzymes from tobacco and other crop plants. Keywords: Transgenic; lysozyme; recombinant protein; antimicrobial; Nicotiana tabacum

Gas chromatographic (GC) profiles of tobacco volatiles and analyses of reducing sugar and total alkaloids as well as subjective smoking evaluations were used to test different tobaccos for tobacco flavor and aroma. Twenty of the major peaks from the GC profiles and subjective smoking scores were analyzed by multiple linear regression. Seven compounds of 20 were included in the regression model that was significant with correlation coefficient R 2 =0.702. Total volatile data for the 20 peaks and the subjective scores were transformed to comparable scales and analyzed by linear regression

Corn seedling xylem exudate has a pH of 5.30 ± 0.05 due to the presence of 10 millimolar malate which has a pK a of 5.13. This concentration of malate tends to buffer the xylem fluid at this pH. Exogenous treatment of corn seedling roots with CaCl 2 caused a concentration-dependent decrease in the pH of the xylem fluid as well as a decrease in the volume of fluid secreted into the xylem. Exogenous 50 millimolar CaCl 2 decreased exudate volume to 8% of control within 0.5 hour. Nitrate and malate deposition into the xylem was prevented by pretreatment in 5 millimolar CaCl 2 ; nitrate deposition was resumed shortly after resumption of malate deposition. Fifty millimolar l-tartrate, an inhibitor of the Cl − , malate-activated ATPase ( in vitro ) of the tonoplast, also decreased exudate volume as well as slightly lowering exudate pH. The osmolality of the exudate was found to be constant at 70 ± 11 milliosmomoles per kilogram in all treatments. Dixon plots (log of exudation rate versus pH of exudate) indicated a pK a of 5.11 for the exudation process which is very close to the pK a of l-malate (5.13). In addition, a Dixon plot of the l-glutamine deposition (l-glutamine being the major form of reduced nitrogen in the exudate) versus pH also indicated a pK a near 5.15. The pH optimum for glutamine transport into the xylem was 5.5. Deposition of glutamine into the xylem may be regulated by the xylem pH (5.30 ± 0.05) which in turn may be regulated by the presence of 10 millimolar malate. It is proposed that the transport of glutamine into the xylem may provide the driving force for the exudation process.

Summary Ultrastructural observations of extranuclear temperature-sensitive induced lethality in a mutant of Nicotiana tabacum L. have shown that chloroplasts in this mutant gradually degenerate in plants grown at reduced temperatures, while mitochondria remain intact (Nessler and Wernsman, 1979). To further elucidate the organellar site of temperature sensitivity in this same tobacco mutant, leaf chlorophyll content, rates of oxygen exchange and ribulose 1,5 bisphosphate carboxylase (RuBPCase) activities were examined in temperature sensitive and control genotypes at varying time intervals during 35 days exposure to lethal temperatures (13°C day/8°C night). Rates of photosynthetic oxygen evolution by leaf disks rapidly declined in mutant plants after one week at low temperatures and remained significantly lower than rates in controls. Total chlorophylls in both mutant and control plants gradually decreased with the increased time at reduced temperatures. This decrease in chlorophyll content of temperature sensitive plants was not sufficiently rapid to account for the sharp drop observed in photosynthetic rates and probably resulted from the disruption of chloroplast membranes. Mitochondrial function in mutant plants was apparently unaffected by prolonged exposure to lethal temperatures as evidenced by the almost constant rates of respiratory oxygen uptake seen throughout the experiment. RuBPCase activities on a leaf area basis were similar in mutant and control plants over the last four sampling dates (days 14–35); thus this enzyme does not appear to be the site of the temperature-sensitive lesion. The loss of chlorophylls, rapid decline in photosynthetic oxygen evolution and continued respiratory oxygen uptake reported here for mutant plants grown at lethal temperatures supports the concept that the chloroplast is the site of extranuclear temperature-sensitivity in this tobacco genotype.