Detection, assay, and preliminary purification of the pigment controlling photoresponsive development of plants.

TLDR: The presence of the photoreversible pigment in intact tissue has been demonstrated with a recording, single-beam spectrophotometer and the system response is sufficiently reproducible that valid difference spectra can be obtained by subtracting one recorded curve from another.

Abstract: Responses of plant materials to radiation indicate that flowering and many other aspects of development are controlled by a reversible photoreaction1'2 involving two forms of a pigment, with action maxima near 660 and 735 mg. The photoreversible pigment can readily be changed from one to the other form, as indicated by response of the plant to irradiation in the region of the appropriate action maximum. Because the nature of the enzymatic action involved is still unknown, it appeared that direct observation of the pigment in the living material and an assay for its isolation would have to be based on spectrophotometric methods. The pigment should show a change in absorption at 655 and 735 mjA following conversion by radiation. The location of the pigment and its concentration in specific cells are evidenced by the photoinduced formation of anthocyanin, which depends upon energy transfer from both forms of the excited pigment.3 The concentration based on a molar absorptivity of 1 X 105 is estimated to be the order of 10-6 molar in the most effective cells and probably about 10-7 molar in the average tissue.4 A spectrophotometer suitable for detecting this low concentration of the pigment in tissue must measure absorption of radiation with high sensitivity in dense light-scattering material. Such measurements cannot be made with commercial instruments. Instrumentation.-The presence of the photoreversible pigment in intact tissue has been demonstrated with a recording, single-beam spectrophotometer. This spectrophotometer5 employs an end-window multiplier-type phototube placed directly behind the sample to collect a large fraction of the transmitted light. The sample is illuminated by monochromatic light from the exit slit of a double, prism monochromator. Spectral measurements can be made on light-scattering samples having optical densities between 0 and 6, with a sensitivity as high as 0.1 for full scale deflection. The noise level is equivalent to an optical density change of 0.002 for samples having an optical density less than 4. Since this is a single-beam instrument the recorded curve includes the spectral response of the instrument in addition to the absorption characteristics of the sample. The system response is sufficiently reproducible that valid difference spectra can be obtained by subtracting one recorded curve from another. When the spectral curve is very steep in the region of interest, an electrical compensation can be applied to alter the slope of the curve. This is achieved with a potentiometer geared to the wavelength drum which supplies an additional signal to the Y-axis. The compensation merely alters the system response to make it easier to compute difference spectra. A more useful instrument for assay of the pigment is one which measures directly the optical density difference between two fixed wavelengths. Such an instrument,