Date of Award:

5-1969

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Plants, Soils, and Climate

Department name when degree awarded

Plant Nutrition and Biochemistry

Committee Chair(s)

George W. Welkie

Committee

George W. Welkie

Committee

W. S. Boyle

Committee

Herman H. Wiebe

Committee

Gene W. Miller

Committee

M. Coburn Williams

Abstract

Changes in concentrations of pipecolic acid and other free amino acids in Nicotiana tabacum, L. cv. Xanthi induced by tobacco mosaic virus (TMV) incubated at 75 or 95 F after inoculation were investigated gas chromatographically. A preliminary extraction and gas chromatographic technique for separation and quantitative determination of the free amino acids, using hydroxyproline as an internal standard, was developed.

Amino acids were converted into their trifluoroacetyl n-butyl (TAB) derivatives and gas chromatographed on 0.325 percent ethylene glycol adipate (EGA) columns. Resolution was generally good, but glycine-isoleucine and phenylalanine-aspartic acids were not resolved on old columns. The over-all recovery of amino acids was at least 100 ± 6.5 percent, and on a relative basis the average recovery was 100 ± 2.2 percent.

Relative molar responses, relative retention times, and relative retention temperatures of 19 TAB amino acid derivatives including pipecolic α-aminoadipic, and azetidine-2-carboxylic acids were reported. Several unknown peaks were present in the chromatograms obtained from plant samples. The major unknowns were thought to be polyphenols or their derivatives.

Plants were incubated at 95 or 75 F for 24, 48, or 72 hours after TMV inoculation, water inoculation (control), or not inoculated (control). Rubbed controls and controls were similar in amino acid content after 72 hours. Rubbed control and TMV infected leaves had higher but similar amino acid leaves after 24 hours. After 48 hours the concentrations in the infected leaves were nearly identical at 75 and 95 F, although pipecolic acid accumulated at 75 F. The 75 and 95 F amino acid levels were similar to the rubbed control levels after 72 hours. Proline and pipecolic acid were up considerably at 75 F.

Systemically infected plants incubated for 48 hours at 95 F were transferred to 75 F for various periods of time to induce rapid necrosis. Within 1 hour wilting began, and after 8 hours the inoculated leaves were shriveled and hanging limply from the stems. Concentrations of most amino acids were down after 1 hour but fell to a fifth of that content at 4 hours and remained low.

Infiltration of DL-lysine HCl plus nutrient solution into detached rubbed control and TMV infected leaves led to wilting but no lesions. Inoculated leaves infiltrated only with nutrient solution developed lesions. Lysine, pipecolic, and α-aminoadipic acid were up 40- to 50-fold in lysine infiltrated leaves.

Results of the study showed that tobacco leaf tissue can catabolize lysine into pipecolic acid and that pipecolic acid is present in milli-micromolar concentrations in tobacco under normal conditions. Pipecolic acid accumulation was probably the result of a secondary process associated with necrosis rather than a cause of necrosis. Necrosis did not correlate with pipecolic, glutamic, or α-aminoadipic acid increases, nor did it correlate with elevated lysine concentrations, unless the increases were very large.

Pipecolic acid accumulation was correlated with polyphenol oxidase activity and quinone accumulation. A mechanism in which an increased polyphenol oxidase activity and quinone accumulation increased the rate of oxidative deamination was suggested as the cause of pipecolic acid accumulation in TMV infected, hypersensitive tobacco.

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